Dna molecules encoding ligand-gated ion channels from drosophila melanogaster

ABSTRACT

The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode  Drosophila melanogaster  ligand-gated ion channel proteins. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding  Drosophila  ligand-gated ion channel proteins, substantially purified forms of associated  Drosophila  ligand-gated ion channel proteins and recombinant membrane fractions comprising these proteins, associated mutant proteins, and methods associated with identifying compounds which modulate associated  Drosophila melanogaster  ligand-gated ion channel proteins, which will be useful as insecticides.

CROSS-REFERENCE TO RELATED APPLICATIONS

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STATEMENT REGARDING FEDERALLY-SPONSORED R&D

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REFERENCE TO MICROFICHE APPENDIX

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FIELD OF THE INVENTION

The present invention relates in part to isolated nucleic acid molecules(polynucleotides) which encode Drosophila melanogaster ligand-gated ionchannels. The present invention also relates to recombinant vectors andrecombinant hosts which contain a DNA fragment encoding Drosophilaligand-gated ion channels, substantially purified forms of associatedDrosophila ligand-gated ion channels and recombinant membrane fractionscomprising these proteins, associated mutant proteins, and methodsassociated with identifying compounds which modulate associatedDrosophila melanogaster ligand-gated ion channels, which will be usefulas insecticides.

BACKGROUND OF THE INVENTION

Glutamate-gated chloride channels, or H-receptors, have been identifiedin arthropod nerve and muscle (Lingle et al, 1981, Brain Res. 212:481-488; Horseman et al., 1988, Neurosci. Lett. 85: 65-70; Wafford andSattelle, 1989, J. Exp. Bio. 144: 449462; Lea and Usherwood, 1973, Comp.Gen. Parmacol. 4: 333-350; and Cull-Candy, 1976, J. Physiol. 255:449-464).

Invertebrate glutamate-gated chloride channels are important targets forthe widely used avermectin class of anthelmintic and insecticidalcompounds. The avermectins are a family of macrocyclic lactonesoriginally isolated from the actinomycete Streptomyces avermitilis. Thesemisynthetic avermectin derivative, ivermectin(22,23-dihydro-avermectin B_(1a)), is used throughout the world to treatparasitic helminths and insect pests of man and animals. The avermectinsremain the most potent broad spectrum endectocides exhibiting lowtoxicity to the host. After many years of use in the field, thereremains little resistance to avermectin in the insect population. Thecombination of good therapeutic index and low resistance stronglysuggests that the glutamate-gated chloride (GluCl) channels remain goodtargets for insecticide development.

Glutamate-gated chloride channels have been cloned from the soilnematode Caenorhabditis elegans (Cully et al., 1994, Nature 371:707-711; see also U.S. Pat. No. 5,527,703 and Arena et al., 1992,Molecular Brain Research. 15: 339-348) and Ctenocephalides felis (flea;see WO 99/07828).

In addition, a gene encoding a glutamate-gated chloride channel fromDrosophila melanogaster was previously identified (Cully et al., 1996,J. Biol. Chem. 271: 20187-20191; see also U.S. Pat. No.5,693,492).

O'Tousa et al. (1989, J. Neurogenetics 6: 41-52) map photoreceptormutations to the ChIII 92B region of the Droshphila genome.

Stuart, 1999, Neuron 22:431433 reviews the art which suggests thathistamine is an invertrbrate nuerotransmitter.

Despite the identification of the aforementioned cDNA clones encodingGluCls, including a previous identification of a Drosophila GluCl gene(see U.S. Pat. No. 5,693,492), it would be advantageous to identifyadditional genes which encode invertebrate ligand-gated ion channels,including but not limited to additional GluCls or other ligand-gatedchannels, such as a ligand-gated ion channel (LGIC) which is activatedby histamine, which may provide additional targets for effectiveinsecticides, in turn allowing for improved screening to identify novelLGIC modulators that may have insecticidal, mitacidal and/or nematocidalactivity for animal health or crop protection. The present inventionaddresses and meets these needs by disclosing novel genes which expressa Drosophila melanogaster ligand-gated ion channel, wherein expressionof the respective Drosophila gene in Xenopus oocytes or otherappropriate host cell results in an active LGIC. Heterologous expressionof a respective LGIC(s) of the present invention will allow thepharmacological analysis of compounds active against parasiticinvertebrate species relevant to animal and human health. Such speciesinclude worms, fleas, tick, and lice. Heterologous cell lines expressingan active LGIC can be used to establish functional or binding assays toidentify novel LGIC modulators that may be useful in control of theaforementioned species groups.

SUMMARY OF THE INVENTION

The present invention relates to an isolated or purified nucleic acidmolecule (polynucleotide) which encodes a novel Drosophila melanogasterinvertebrate ligand-gated ion channel (LGIC) protein which comprises atleast a portion of a LGIC receptor. The DNA molecules disclosed hereinmay be transfected into a host cell of choice wherein the recombinanthost cell provides a source for substantial levels of an expressedfunctional single, homomultimer or heteromultimer LGIC channel. The cDNAclones described herein express a functional single channel protein,both of which are activated by histamine. Therefore, these DmLGICchannels form receptors which provide for additional screening targetsto identify modulators of these channels, modulators which may act aseffective insecticidal, mitacidal and/or nematocidal treatment for usein animal and human health and/or crop protection.

The present invention further relates to an isolated nucleic acidmolecule (polynucleotide) which encodes mRNA which expresses a novelDrosophila melanogaster LGIC protein, this DNA molecule comprising thenucleotide sequence disclosed herein as SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5 and SEQ ID NO:6.

The present invention also relates to biologically active fragments ormutants of SEQ ID NOs: 1, 3, 5 and 6 which encode mRNA expressing anovel Drosophila melanogaster invertebrate LGIC protein. Any suchbiologically active fragment and/or mutant will encode either a proteinor protein fragment which at least substantially mimics thepharmacological properties of the respective Drosophila LGIC protein,including but not limited to the Drosophila LGIC proteins as set forthin SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:7. Any such polynucleotideincludes but is not necessarily limited to nucleotide substitutions,deletions, additions, amino-terminal truncations and carboxy-terminaltruncations such that these mutations encode mRNA which express afunctional Drosophila LGIC in a eukaryotic cell, such as Xenopusoocytes, so as to be useful for screening for agonists and/orantagonists of Drosophila LGIC activity.

A preferred aspect of this portion of the present invention is disclosedin FIG. 1 (SEQ ID NO:1; designated AC05-10), FIG. 3 (SEQ ID NO:3;designated AC05-11), FIG. 5 (SEQ ID NO:5; designated AC15-4) and FIG. 6(SEQ ID NO:6) encoding a novel Drosophila melanogaster LGIC protein.

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification.

The present invention also relates to a substantially purified form of aDrosophila LGIC protein, which comprises the amino acid sequencedisclosed in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) and FIG. 7 (SEQID NO:7).

A preferred aspect of this portion of the present invention is aDrosophila LGIC protein which consists of the amino acid sequencedisclosed in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) and FIG. 7 (SEQID NO:7).

Another preferred aspect of the present invention relates to asubstantially purified, fully processed (including proteolyticprocessing, glycosylation and/or phosphorylation), mature LGIC proteinobtained from a recombinant host cell containing a DNA expression vectorcomprising nucleotide sequence as set forth in SEQ ID NOs: 1, 3, 5and/or 6 which express the respective DmLGIC protein. It is especiallypreferred is that the recombinant host cell be a eukaryotic host cell,such as a mammalian cell line, or Xenopus oocytes, as noted above.

Another preferred aspect of the present invention relates to asubstantially purified membrane preparation, partially purified membranepreparation, or cell lysate which has been obtained from a recombinanthost cell transformed or transfected with a DNA expression vector whichcomprises and appropriately expresses a complete open reading frame asset forth in SEQ ID NOs: 1, 3, 5 and/or 6, resulting in a functionalform of the respective DmLGIC. The subcellular membrane fractions and/ormembrane-containing cell lysates from the recombinant host cells (bothprokaryotic and eukaryotic as well as both stably and transientlytransformed cells) contain the functional and processed proteins encodedby the nucleic acids of the present invention. This recombinant-basedmembrane preparation may comprise a Drosophila LGIC and is essentiallyfree from contaminating proteins, including but not limited to otherDrosophila source proteins or host proteins from a recombinant cellwhich expresses the AC05-10 (SEQ ID NO:2), AC05-11 (SEQ ID NO:4) and/orAC154/AC15-25 (SEQ ID NO:7) LGIC protein. Therefore, a preferred aspectof the invention is a membrane preparation which contains a DrosophilaLGIC comprising the functional form of the full length LGIC proteins asdisclosed in FIG. 2 (SEQ ID NO:2, AC05-10), FIG. 4 (SEQ ID NO:4, AC05-11), and FIG. 7 (SEQ ID NO:7, AC15-4/AC14-25). These subcellular membranefractions will comprise either wild type and/or mutant variations whichare biologically functional forms of the Drosophila LGIC (including butnot limited to functional channels generated by a single polypeptide, orany homomultimer or heteromultimer channel combinations thereof) atlevels substantially above endogenous levels. Any such channel will beuseful in various assays described throughout this specification toselect for modulators of the respective LGIC channel. A preferredeukaryotic host cell of choice to express the LGICs of the presentinvention is a mammalian cell line, or Xenopus oocytes.

The present invention also relates to biologically active fragmentsand/or mutants of an Drosophila LGIC protein, comprising the amino acidsequence as set forth in SEQ ID NOs: 2, 4 and/or 7, including but notnecessarily limited to amino acid substitutions, deletions, additions,amino terminal truncations and carboxy-terminal truncations such thatthese mutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening forselective modulators, including but not limited to agonists and/orantagonists for Drosophila ligand-gated ion channel pharmacology.

A preferred aspect of the present invention is disclosed in FIG. 2 (SEQID NO:2), FIG. 4 (SEQ ID NO:4) and FIG. 7 (SEQ ID NO:7), respectiveamino acid sequences which compose the Drosophila LGIC proteins of thepresent invention. Characterization of one or more of these channelproteins allows for screening to identify novel LGIC modulators that mayhave insecticidal, mitacidal and/or nematocidal activity for animalhealth or crop protection. As noted above, heterologous expression offunctional single channel, homomultimer and/or heteromultimercombination of Drosophila melanogaster LGICs disclosed herein iscontemplated at levels substantially above endogenous levels and willallow for the pharmacological analysis of compounds active againstparasitic invertebrate species relevant to animal and human health. Suchspecies include worms, fleas, tick, and lice. Heterologous cell linesexpressing a functional DmLGIC channel (e.g., functional forms of SEQ IDNOs: 2, 4 and/or 7), can be used to establish functional or bindingassays to identify novel LGIC modulators that may be useful in controlof the aforementioned species groups.

The present invention also relates to polyclonal and monoclonalantibodies raised against forms of DmLGIC, or a biologically activefragment thereof.

The present invention also relates to DmLGIC fusion constructs,including but not limited to fusion constructs which express a portionof the DmLGIC linked to various markers, including but in no way limitedto GFP (Green fluorescent protein), the MYC epitope, GST, and Fc. Anysuch fusion constructs may be expressed in the cell line of interest andused to screen for modulators of one or more of the DmLGIC proteinsdisclosed herein.

The present invention relates to methods of expressing Drosophila LGICproteins and biological equivalents disclosed herein, assays employingthese gene products, recombinant host cells which comprise DNAconstructs which express these proteins, and compounds identifiedthrough these assays which act as agonists or antagonists of LGICactivity.

It is an object of the present invention to provide an isolated nucleicacid molecule (e.g., SEQ ID NOs: 1, 3, 5 and 6) which encodes a novelform of Drosophila LGIC, or fragments, mutants or derivatives DmLGIC, asset forth in SEQ ID NOs: 2, 4, and 7, respectively. Any suchpolynucleotide includes but is not necessarily limited to nucleotidesubstitutions, deletions, additions, amino-terminal truncations andcarboxy-terminal truncations such that these mutations encode MRNA whichexpress a protein or protein fragment of diagnostic, therapeutic orprophylactic use and would be useful for screening for selectivemodulators for invertebrate ligand-gated ion channel pharmacology.

It is a further object of the present invention to provide theDrosophila LGIC proteins or protein fragments encoded by the nucleicacid molecules referred to in the preceding paragraph.

It is a further object of the present invention to provide recombinantvectors and recombinant host cells which comprise a nucleic acidsequence encoding Drosophila LGIC proteins or a biological equivalentthereof.

It is an object of the present invention to provide a substantiallypurified form of Drosophila LGIC proteins, as set forth in SEQ ID NOs:2, 4, and 7.

Is another object of the present invention to provide a substantiallypurified recombinant form of a Drosophila LGIC protein which has beenobtained from a recombinant host cell transformed or transfected with aDNA expression vector which comprises and appropriately expresses acomplete open reading frame as set forth in SEQ ID NOs: 1, 3, 5 and 6,resulting in a functional, processed form of the respective DmLGIC. Itis especially preferred is that the recombinant host cell be aeukaryotic host cell, such as a mammalian cell line.

It is an object of the present invention to provide for biologicallyactive fragments and/or mutants of Drosophila LGIC proteins, such as setforth in SEQ ID NOs: 2, 4, and 7, including but not necessarily limitedto amino acid substitutions, deletions, additions, amino terminaltruncations and carboxy-terminal truncations such that these mutationsprovide for proteins or protein fragments of diagnostic, therapeuticand/or prophylactic use.

It is further an object of the present invention to provide forsubstantially purified subcellular membrane preparations, partiallypurified subcellular membrane preparations, or crude lysates fromrecombinant cells which comprise pharmacologically active DrosophilaLGICs, especially subcellular fractions obtained from a host celltransfected or transformed with a DNA vector comprising a nucleotidesequence which encodes a protein which comprises the amino acid as setforth in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) and/or FIG. 7 (SEQID NO:7).

It is another object of the present invention to provide a substantiallypurified membrane preparation, partially purified subcellular membranepreparations, and/or crude lysates obtained from a recombinant host celltransformed or transfected with a DNA expression vector which comprisesand appropriately expresses a complete open reading frame as set forthin SEQ ID NOs: 1, 3, 5, and/or 6, resulting in a functional, processedform of the respective DmLGIC. It is especially preferred is that therecombinant host cell be a eukaryotic host cell, such as a mammaliancell line, or Xenopus oocytes.

It is also an object of the present invention to use Drosophila LGICproteins or membrane preparations containing Drosophila LGIC proteins ora biological equivalent to screen for modulators, preferably selectivemodulators, of Drosophila LGIC activity. Any such protein or membraneassociated protein may be useful in screening and selecting thesemodulators active against parasitic invertebrate species relevant toanimal and human health. Such species include worms, fleas, tick, andlice. These membrane preparations may be generated from heterologouscell lines expressing these LGICs and may constitute full lengthprotein, biologically active fragments of the full length protein or mayrely on fusion proteins expressed from various fusion constructs whichmay be constructed with materials available in the art.

As used herein, “substantially free from other nucleic acids” means atleast 90%, preferably 95%, more preferably 99%, and even more preferably99.9%, free of other nucleic acids. As used interchangeably with theterms “substantially free from other nucleic acids” or “substantiallypurified” or “isolated nucleic acid” or “purified nucleic acid” alsorefer to a DNA molecules which comprises a coding region for aDrosophila LGIC protein that has been purified away from other cellularcomponents. Thus, a Drosophila LGIC DNA preparation that issubstantially free from other nucleic acids will contain, as a percentof its total nucleic acid, no more than 10%, preferably no more than 5%,more preferably no more than 1%, and even more preferably no more than0.1%, of non-Drosophila LGIC nucleic acids. Whether a given DrosophilaLGIC DNA preparation is substantially free from other nucleic acids canbe determined by such conventional techniques of assessing nucleic acidpurity as, e.g., agarose gel electrophoresis combined with appropriatestaining methods, e.g., ethidium bromide staining, or by sequencing.

As used herein, “substantially free from other proteins” or“substantially purified” means at least 90%, preferably 95%, morepreferably 99%, and even more preferably 99.9%, free of other proteins.Thus, a Drosophila LGIC protein preparation that is substantially freefrom other proteins will contain, as a percent of its total protein, nomore than 10%, preferably no more than 5%, more preferably no more than1%, and even more preferably no more than 0.1%, of non-Drosophila LGICproteins. Whether a given Drosophila LGIC protein preparation issubstantially free from other proteins can be determined by suchconventional techniques of assessing protein purity as, e.g., sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combinedwith appropriate detection methods, e.g., silver staining orimmunoblotting. As used interchangeably with the terms “substantiallyfree from other proteins” or “substantially purified”, the terms“isolated Drosophila LGIC protein” or “purified Drosophila LGIC protein”also refer to Drosophila LGIC protein that has been isolated from anatural source. Use of the term “isolated” or “purified” indicates thatDrosophila LGIC protein has been removed from its normal cellularenvironment. Thus, an isolated Drosophila LGIC protein may be in acell-free solution or placed in a different cellular environment fromthat in which it occurs naturally. The term isolated does not imply thatan isolated Drosophila LGIC protein is the only protein present, butinstead means that an isolated Drosophila LGIC protein is substantiallyfree of other proteins and non-amino acid material (e.g., nucleic acids,lipids, carbohydrates) naturally associated with the Drosophila LGICprotein in vivo. Thus, a Drosophila LGIC protein that is recombinantlyexpressed in a prokaryotic or eukaryotic cell and substantially purifiedfrom this host cell which does not naturally (i.e., withoutintervention) express this LGIC protein is of course “isolatedDrosophila LGIC protein” under any circumstances referred to herein. Asnoted above, a Drosophila LGIC protein preparation that is an isolatedor purified Drosophila LGIC protein will be substantially free fromother proteins will contain, as a percent of its total protein, no morethan 10%, preferably no more than 5%, more preferably no more than 1%,and even more preferably no more than 0. 1%, of non-Drosophila LGICproteins.

As used interchangeably herein, “functional equivalent” or “biologicallyactive equivalent” means a protein which does not have exactly the sameamino acid sequence as naturally occurring Drosophila LGIC, due toalternative splicing, deletions, mutations, substitutions, or additions,but retains substantially the same biological activity as DrosophilaLGIC. Such functional equivalents will have significant amino acidsequence identity with naturally occurring Drosophila LGIC and genes andcDNA encoding such functional equivalents can be detected by reducedstringency hybridization with a DNA sequence encoding naturallyoccurring Drosophila LGIC. For example, a naturally occurring DrosophilaLGIC disclosed herein comprises the amino acid sequence shown as SEQ IDNO:2 and is encoded by SEQ ID NO: 1. A nucleic acid encoding afunctional equivalent has at least about 50% identity at the nucleotidelevel to SEQ ID NO: 1.

As used herein, “a conservative amino acid substitution” refers to thereplacement of one amino acid residue by another, chemically similar,amino acid residue. Examples of such conservative substitutions are:substitution of one hydrophobic residue (isoleucine, leucine, valine, ormethionine) for another; substitution of one polar residue for anotherpolar residue of the same charge (e.g., arginine for lysine; glutamicacid for aspartic acid).

As used herein, “LGIC” refers to a-ligand-gated ion channel-

As used herein, “DmLGIC” refers to a-Drosophila melanogasterligand-gated ion channel-.

As used herein, “GluCl” refers to-L-glutamate gated chloride channel-.

As used herein, “DmGluCl” refers to-Drosophila melanogaster L-glutamategated chloride channel-.

As used herein, the term “mammalian” will refer to any mammal, includinga human being.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence which of the Drosophila LGIC clone,AC05-10, as set forth in SEQ ID NO:1.

FIG. 2 shows the amino acid sequence of the Drosophila LGIC AC05-10protein, as set forth in SEQ ID NO:2.

FIG. 3 shows the nucleotide sequence which of the Drosophila LGIC clone,AC05-11, as set forth in SEQ ID NO:3.

FIG. 4 shows the amino acid sequence of the Drosophila LGIC AC05-11protein, as set forth in SEQ ID NO:4.

FIG. 5 shows the nucleotide sequence which of the Drosophila LGIC clone,AC154, as set forth in SEQ ID NO:5.

FIG. 6 shows the nucleotide sequence which of the Drosophila LGIC clone,AC15-25, as set forth in SEQ ID NO:6.

FIG. 7 shows the amino acid sequence of the Drosophila LGICAC154/AC15-25 protein, as set forth in SEQ ID NO:7.

FIG. 8 shows the activation of a recombinant DmLGIC (AC05-10) byhistamine in transfected Xenopus oocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes a Drosophila melanogaster invertebrateLGIC protein, which are phylogentically related to known DmGluClproteins but which show alternative pharmacology, and hence, representnovel insecticide targets. The isolated or purified nucleic acidmolecules of the present invention are substantially free from othernucleic acids. For most cloning purposes, DNA is a preferred nucleicacid. As noted above, the DNA molecules disclosed herein it may betransfected into a host cell of choice wherein the recombinant host cellprovides a source for substantial levels of an expressed functionalsingle, homomultimer or heteromultimer LGIC channel. The cDNA clonesdescribed herein express a functional single channel protein, both ofwhich are activated by histamine. Therefore, these DmLGIC channels formreceptors which provide for additional screening targets to identifymodulators of these channels, modulators which may act as effectiveinsecticidal, mitacidal and/or nematocidal treatment for use in animaland human health and/or crop protection. The DNA molecules disclosedherein are transfected into a host cell of choice wherein therecombinant host cell provides a source for substantial levels of anexpressed functional single, homomultimer or heteromultimer LGICchannel.

The present invention relates to an isolated nucleic acid molecule(polynucleotide) which encodes mRNA which expresses a novel Drosophilamelanogaster invertebrate LGIC protein, this DNA molecule comprising thenucleotide sequence disclosed herein as SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, and SEQ ID NO:6. The isolation and characterization of the DmLGICnucleic acid molecules of the present invention were identified asdescribed in detail in Example Section 1.

Invertebrate glutamate-gated chloride channels (GluCls) are related tothe glycine- and GABA-gated chloride channels and are distinct from theexcitatory glutamate receptors (e.g. NMDA or AMPA receptors). The firsttwo members of the GluCl family were identified in the nematode C.elegans, following a functional screen for the receptor of theanthelmintic drug ivermectin. Several additional GluCls have now beencloned in other invertebrate species. However, there is no evidence yetfor GluCl counterparts in vertebrates; because of this, GluCls and anyrelated ligand-gated channels are potentially excellent targets foranthelmintics, insecticides, acaricides, etc. Specific GluCl modulators,such as nodulisporic acid and its derivatives, indeed have an idealsafety profile because they lack mechanism-based toxicity invertebrates. The present invention relates in part to four novelDrosophila LGIC clones, AC05-10, AC05-11, AC154 and AC15-25, which showhomology to the earlier identified DmGluCla and to C. felis CfGluCl DNA.

The present invention relates to the isolated or purified DNA moleculedescribed in FIG. 1 (AC05-10) and set forth as SEQ ID NO:1, whichencodes the Drosophila LGIC protein described in FIG. 2 and set forth asSEQ ID NO:2, the nucleotide sequence of AC05-10 is as follows:CAATCGTCGC GATAACTCTG CCGTTTCTTT ATTGGTTTTT GCTGCGCGAC GAGTAAAATA (SEQID NO:1) TAATTCCTCG CTTACTAATC CTCCGAGCAA GTTCATTCTC AAGCGCACCCAGAGATGAGC TACTTTGGGA ATTGACATGG ACTGCGGAGC AATGAGTGCC AGAGGAACAATATCAAAGCC GAAGGTAGTG TGTTCATA AT   G CAAAGCCCA ACTAGCAAAT TGGTAGAATTCAGGTGCCTT ATTGCGTTGG CAATATATTT GCACGCGCTG GAGCAATCGA TCCAGCACTGCCATTGTGTT CATGGTTACA GAAATAACAC GGAGAGCGCC GAGCTGGTCT CCCACTACGAGTCGAGTCTT TCGCTCCCGG ACATTTTGCC CATTCCCTCA AAGACGTACG ACAAGAACCGGGCTCCCAAG CTCCTCGGCC AGCCCACAGT AGTCTACTTC CATGTCACGG TCCTCTCCCTGGACTCCATT AACGAGGAGT CTATGACCTA TGTGACGGAC ATCTTCCTTG CACAAAGCTGGCGTGATCCT CGCCTGCGGT TGCCTGAGAA CATGAGTGAG CAGTATCGCA TATTGGATGTCGACTGGTTG CACAGCATTT GGCGGCCCGA TTGCTTCTTT AAGAACGCCA AAAAGGTCACCTTCCATGAG ATGAGCATTC CCAAGCACTA TCTCTGGCTG TACCACGACA AAACGCTGCTCTATATGTCC AAACTCACGT TGGTCCTGTC GTGCGCCATG AAGTTTGAGT CCTATCCGCATGACACGCAA ATCTGCTCCA TGATGATCGA GAGTTTATCC CATACGGTGG AAGATTTGGTTTTCATTTGG AACATGACCG ACCCACTTGT GGTTAACACG GAGATTGAGT TGCCGCAGCTAGACATATCA AATAACTACA CAACCGACTG TACTATAGAG TACTCAACAG GTAACTTCACCTGCCTGGCC ATTGTGTTCA ACCTGCGCCG ACGCCTGGGT TACCATTTGT TCCACACCTACATCCCCTCG GCTCTGATTG TGGTCATGTC TTGGATATCG TTTTGGATAA AACCAGAAGCGATACCCGCC CGTGTAACTC TGGGAGTGAC CTCACTGCTA ACCCTGGCCA CCCAGAATACCCAGTCGCAA CAATCGCTGC CGCCGGTTTC GTATGTCAAG GCTATAGACG TCTGGATGTCGTCCTGTTCG GTGTTTGTAT TCCTTTCTCT GATGGAATTT GCAGTGGTCA ACAATTTTATGGGACCGGTG GCCACAAAGG CAATGAAGGG GTATTCGGAC GAGAACATCA GTGATCTGGACGACCTAAAG TCTGCACTAC AGCATCATCG GGAATCGATT ATTGAGCCCC AGTACGACACTTTCTGCCAT GGCCATGCCA CAGCCATTTA TATAGACAAA TTCTCGCGCT TTTTCTTCCCGTTTTCGTTC TTTATACTCA ATATTGTCTA TTGGACAACG TTCCTA TGA T GGATGGAAAAGTTTCTCCGA AGGAATAGAG CGTAAACA.

The present invention also relates to the isolated or purified DNAmolecule described in FIG. 3 (AC05-1 1) and set forth as SEQ ID NO:3,which encodes the Drosophila LGIC protein described in FIG. 4 and setforth as SEQ ID NO:4, the nucleotide sequence AC05-11 as follows:CAATCGTCGC GATAACTCTG CCGTTTCTTT ATTGGTTTTT GCTGCGCGAC GAGTAAAATA (SEQID NO:3) TAATTCCTCG CTTACTAATC CTCCGAGCAA GTTCATTCTC AAGCGCACCCAGAGATGAGC TACTTTGGGA ATTGACATGG ACTGCGGAGC AATGAGTGCC AGAGGAACAATATCAAAGCC GAAGGTAGTG TGTTCATA AT   G CAAAGCCCA ACTAGCAAAT TGGTAGAATTCAGGTGCCTT ATTGCGTTGG CAATATATTT GCACGCGCTG GAGCAATCGA TCCAGCACTGCCATTGTGTT CATGGTTACA GAAATAACAC GGAGAGCGCC GAGCTGGTCT CCCACTACGAGTCGAGTCTT TCGCTCCCGG ACATTTTGCC CATTCCCTCA AAGACGTACG ACAAGAACCGGGCTCCCAAG CTCCTCGGCC AGCCCACAGT AGTCTACTTC CATGTCACGG TCCTCTCCCTGGACTCCATT AACGAGGAGT CTATGACCTA TGTGACGGAC ATCTTCCTTG CACAAAGCTGGCGTGATCCT CGCCTGCGGT TGCCTGAGAA CATGAGTGAG CAGTATCGCA TATTGGATGTCGACTGGTTG CACAGCATTT GGCGGCCCGA TTGCTTCTTT AAGAACGCCA AAAAGGTCACCTTCCATGAG ATGAGCATTC CCAATCACTA TCTCTGGCTG TACCACGACA AAACGCTGCTCTATATGTCC AAACTCACGT TGGTCCTGTC GTGCGCCATG AAGTTTGAGT CCTATCCGCATGACACGCAA ATCTGCTCCA TGATGATCGA GAGTTTATCC CATACGGTGG AAGATTTGGTTTTCATTTGG AACATGACCG ACCCACTTGT GGTTAACACG GAGATTGAGT TGCCGCAGCTAGACATATCA AATAACTACA CAACCGACTG TACTATAGAG TACTCAACAG GTAACTTCACCTGCCTGGCC ATTGTGTTCA ACCTGCGCCG ACGCCTGGGT TACCATTTGT TCCACACCTACATCCCCTCG ATTGTGTTCA ACCTGCGCCG ACGCCTGGGT TACCATTTGT TCCACACCTACATCCCCTCG GCTCTGATTG TGGTCATGTC TTGGATATCG TTTTGGATAA AACCAGAAGCGATACCCGCC CGTGTAACTC TGGGAGTGAC CTCACTGCTA ACCCTGGCCA CCCAGAATACCCAGTCGCAA CAATCGCTGC CGCCGGTTTC GTATGTCAAG GCTATAGACG TCTGGATGTCGTCCTGTTCG GTGTTTGTAT TCCTTTCTCT GATGGAATTT GCAGTGGTCA ACAATTTTATGGGACCGGTG GCCACAAAGG CAATGAAGGG GTATTCGGAC GAGAACATCA GTGATCTGGACGACCTAAAG CATCATCGGG AATCGATTAT TGAGCCCCAG TACGACACTT TCTGCCATGGCCATGCCACA GCCATTTATA TAGACAAATT CTCGCGCTTT TTCTTCCCGT TTTCGTTCTTTATACTCAAT ATTGTCTATT GGACAACGTT CCTA TGA TGG ATGGAAAAGT TTCTCCGAAGGAATAGAGCG TAAACA.

The present invention also relates to the isolated or purified DNAmolecule described in FIG. 5 (AC154) and set forth as SEQ ID NO:5, whichencodes the Drosophila LGIC protein described in FIG. 7 and set forth asSEQ ID NO:7, the nucleotide sequence AC154 as follows: AACTGCCAAGACGTTTAGAA CGGAAAAACT GAATTTTCAA AAATATTTCG AGTAAACTGT (SEQ ID NO:5)TAAATGCTGA AGTGTTCTGA AATATTCCTT AAAACATAGA AACCTTCTTT GACATCTTTATAAAGCAATA AAATTCATTC GGGAAGTTTA TGAATAGTGG TGTTATTAAT CATGCCATTTGTGGCGTCAA GCTGATGGTT ATGTAATCTC TGTGAAGATT CTAGAAATCC AACAGAAATATATTGCTTCG AAAACCAAGC AAAGATTACT TGACTGGAGA GGAAAGCTAT TTCGAATTCATCTAAAAACT GTAAAGAGTT CACATTAAA A   TG GTGTTCCA AATAATAATC CTGGTGATCTGCACCATCTG CATGAAGCAC TACGCCAAAG GGGAGTTTCA ACAAAGTCTG GCCATAACCGACATCCTGCC CGAGGACATC AAGCGTTACG ACAAGATGAG ACCGCCGAAG AAAGAGGGTCAGCCGACGAT AGTCTACTTC CATGTGACTG TGATGGGTCT GGACTCCATT GATGAGAACTCGATGACTTA TGTGGCGGAT GTGTTCTTTG CACAGACGTG GAAGGATCAT CGCCTGCGATTGCCGGAGAA TATGACACAG GAATACCGCC TGCTCGAGGT GGACTGGCTA AAAAATATGTGGCGCCCGGA TTCGTTTTTC AAAAACGCCA AATCGGTGAC CTTTCAGACC ATGACAATACCCAATCACTA TATGTGGCTG TACAAGGATA AGACCATTCT CTATATGGTC AAGCTAACACTGAAGCTGTC CTGCATCATG AATTTCGCCA TTTATCCTCA TGACACACAG GAGTGCAAGCTGCAAATGGA AAGCCTGTCC CACACCACGG ATGACTTGAT ATTCCAGTGG GATCCAACAACGCCCCTTGT GGTTGATGAA AACATCGAAC TGCCGCAGGT GGCCCTCATC CGGAATGAAACGGCGGACTG CACCCAGGTT TATTCCACTG GCAACTTCAC ATGCCTGGAG GTGGTGTTCACCCTTAAGCG TCGTTTGGTT TACTACGTTT TCAACACCTA CATTCCCACC TGCATGATAGTGATCATGTC ATGGGTATCC TTCTGGATCA AACCGGAGGC GGCACCAGCC CGTGTGACTCTGGGTGTCAC CTCCTTGCTA ACGCTTTCCA CGCAACACGC CAAATCGCAG TCGTCTTTGCCACCTGTTTC CTATCTCAAG GCAGTGGACG CCTTTATGTC CGTTTGCACG GTGTTCGTGTTTATGGCCCT CATGGAGTAT TGTCTAATAA ACATCGTCCT GAGCGACACG CCCATTCCCAAGCCGATGGC TTATCCACCC AAACCTGTGG CGGGCGATGG GCCCAAGAAA GAGGGCGAGGGTGCTCCTCC TGGGGGCAGC AACTCGACGG CCAGCAAGCA ACAAGCCACC ATGTTGCCACTGGCCGATGA AAAGATCGAG AAAATTGAGA AGATCTTTGA CGAGATGACC AAGAATAGAAGGATTGTAAC CACCACACGC CGCGTGGTGC GTCCACCATT GGACGCCGAT GGTCCGTGGATTCCGCGACA GGAGTCGCGG ATAATACTGA CCCCGACTAT CGCTCCGCCG CCACCGCCCCCTCAGCCAGC GGCACCGGAA CCGGAACTAC CCAAGCCGAA ACTCACACCC GCCCAGGAGCGGCTCAAGCG GGCTATATAT ATAGATCGGT CCTCGCGCGT CCTTTTCCCC GCCCTCTTCGCCAGTCTGAA TGGCATCTAC TGGTGTGTGT TCTACGAGTA TCTA TAA GGA CTACGACGACTGTGCCCTGT AAATACTTTC GCTAGCTCTC TGGCACTCCA TCCGAGTGTT AAACGTTGATTGTTCGCATA TATCGAAACG TGTATCGCAA ATTTAATCTT AAGCTTTCAC GCACAAGCTTTAAGTCAATG AATTTTAAAC ATAGATTATT GTTAAACCAG AAGGAAGGAA TAATGGTACAGATGGAGATC TGATTACAGG ATAAATTACA AATTATCAAT TCAATTCCTA AAATGCTTAAAGTTAATCAA GTGACGTAGT AGCTGATGTA GCC.

The present invention also relates to the isolated or purified DNAmolecule described in FIG. 6 (AC15-25), as set forth as SEQ ID NO:6,which also encodes the Drosophila LGIC protein described in FIG. 7 andset forth as SEQ ID NO:7, the nucleotide sequence AC15-25 as follows:CGAGTAAACT GTTAAATGCT GAAGTGTTCT GAAATATTCC TTAAAACATA GAAACCTTCT (SEQID NO:6) TTGACATCTT TATAAAGCAA TAAAATTCAT TCGGGAAGTT TATGAATAGTGGTGTTATTA ATCATGCCAT TTGTGGCGTC AAGCTGATGG TTATGTAATC TCTGTGAAGATTCTAGAAAT CCAACAGAAA TATATTGCTT CGAAAACCAA GCAAAGATTA CTTGACTGGAGAGGAAAGCT ATTTCGAATT CATCTAAAAA CTGTAGCTCA CATTAAA ATG  GTGTTCCAAATAATAATCCT GGTGATCTGC ACCATCTGCA TGAAGCACTA CGCCAAAGGG GAGTTTCAACAAAGTCTGGC CATAACCGAC ATCCTGCCCG AGGACATCAA GCGTTACGAC AAGATGAGACCGCCGAAGAA AGAGGGTCAG CCGACGATAG TCTACTTCCA TGTGACTGTG ATGGGTCTGGACTCCATTGA TGAGAACTCG ATGACTTATG TGGCGGATGT GTTCTTTGCA CAGACGTGGAAGGATCATCG CCTGCGATTG CCGGAGAATA TGACACAGGA ATACCGCCTG CTCGAGGTGGACTGGCTAAA AAATATGTGG CGGCCGGATT CGTTTTTCAA AAACGCCAAA TCGGTGACCTTTCAGACCAT GACAATACCC AATCACTATA TGTGGCTGTA CAAGGATAAG CAACTTCTGTACATGGTCAA ACTAACACTG AAGCTGTCCT GCATCATGAA CTTCGCCATT TATCCTCATGATACACAGGA GTGCAAGCTG CAAATGGAAA GCCTGTCCCA CACCACGGAT GACTTGATATTTCAGTGGGA TCCAACGACG CCCCTTGTGG TTGATGAAAA CATCGAGCTG CCGCAGGTGGCCCTCATCCG AAATGAAACG GCGGACTGTA CCCAAGTTTA TTCCACTGGC AACTTCACATGCCTGGAGGT GGTGTTCACC CTTAAGCGTC GTTTGGTTTA CTACGTTTTC AACACCTACATTCCCACCTG CATGATAGTG ATCATGTCAT GGGTATCCTT CTGGATCAAA CCGGAGGCGGCACCAGCCCG TGTGACTCTG GGTGTCACCT CCTTGCTAAC GCTTTCCACG CAACACGCCAAATCGCAGTC GTCTTTGCCA CCTGTTTCCT ATCTCAAGGC AGTGGACGCC TTTATGTCCGTTTGCACGGT GTTCGTGTTT ATGGCCCTCA TGGAGTATTG TCTAATAAAC ATCGTCCTGAGCGACACGCC CATTCCCAAG CCGATGGCCT ATCCACCCAA ACCTGTGGCG GGAGATGGGCCCAAGAAAGA GGGCGAGGGT GCTCCTCCTG GGGGCAGCAA CTCGACGGCC AGCAAGCAACAAGCCACCAT GTTGCCACTG GCCGATGAAA AGATCGAGAA AATTGAGAAG ATCTTTGACGAGATGACCAA GAATAGAAGG ATTGTAACCA CCACACGCCG CGTGGTGCGT CCGCCATTGGACGCCGATGG TCCGTGGATT CCGCGACAGG AGTCGCGGAT AATACTGACC CCGACTATCGCTCCGCCGCC ACCGCCCCCT CAGCCAGCGG CACCGGAACC GGAACTGCCC AAGCCGAAACTCACACCCGC CCAGGAGCGG CTCAAGCGGG CTATATATAT AGATCGGTCC TCGCGCGTCCTTTTCCCCGC CCTCTTCGCC AGTCTGAATG GCATCTACTG GTGTGTGTTC TACGAGTATC TA TAAGGACT ACGACGACTG TGCCCTGTAA ATACTTTCGC TAGCTCTCTG GCACTCCATC CGAGTGTTAAACGTTGATTG TTCGCATATA TCGAAACGTG TATCGCAAAT TTAATCTTAA GCTTTCACGCACAAGCTTTA AGTCAATGAA TTTTAAACAT AGATTATTGT TAAACCAGAA GGAAGGAATAATGGTACAGA TGGAGATCTG ATTACAGGAT AAATTACAAA TTATCAATTC AATTCCTAAAATGCTTAAAG TTAATCAAGT GACGTAGTAG CTGATGTAGC CTAAGTGAAT TGTA.

The above-exemplified isolated DNA molecules, shown in FIG. 1, 3 and 5,respectively, comprise the following characteristics:

AC05-10 (SEQ ID NO:1):

1518 nuc. :initiating Met (nuc. 199-201) and “TGA” term. codon(nuc.1477-1479), the open reading frame resuting in an expressed proteinof 426 amino acids, as set forth in SEQ ID NO:2.

AC05-11 (SEQ ID NO:3):

1506 nuc.:initiating Met (nuc. 199-201) and “TGA” term. codon (nuc.1465-1467), the open reading frame resuting in an expressed protein of422 amino acids, as set forth in SEQ ID NO:4.

AC154 (SEQ ID NO:5):

2133 nuc. :initiating Met (nuc. 330-332) and “TAA” term. codon (nuc.1785-1787), the open reading frame resuting in an expressed protein of485 amino acids, as set forth in SEQ ID NO:7.

AC15-25 (SEQ ID NO:6):

2034 nuc. :initiating Met (nuc. 278-280) and “TAA” term. codon (nuc.1733-1735), the open reading frame resulting in an expressed protein of485 amino acids, as set forth in SEQ ID NO:7.

The Ac5-10 and Ac5-11 open reading frames are identical, save for a 12nucleotide insertion within Ac5-10 which encodes a 4 amino acidinsertion within the M3-M4 intracellular loop in Ac05-10 (aSer-Ala-Leu-Gln insertion from amino acid residue 375 through amino acidresidue 378, as set forth in SEQ ID NO:2). Therefore, the AcS-10 proteinis 426 amino acids in length while the Ac5-11 protein is 422 amino acidsin length. Expression of Ac5-10 in Xenopus results in a functional ionchannel that responds to the addition of histamine.

Two clones of Ac15 are disclosed. Ac154 (2073 bp) and Ac15-25 (2034 bp)predict the same protein sequence but differ in 16 silent nucleotidechanges within the 1455 nucleotide open reading frame. The expressedprotein from Ac15-4/Ac15-25 is 485 amino acids in length.

The present invention also relates to biologically active fragments ormutants of SEQ ID NOs:1, 3, 5 and 6 which encode mRNA expressing DmLGIC.Any such biologically active fragment and/or mutant will encode either aprotein or protein fragment which at least substantially mimics the wildtype protein, including but not limited to the wild type forms as setforth in SEQ ID NOs: 2, 4, and/or 7. Any such polynucleotide includesbut is not necessarily limited to nucleotide substitutions, deletions,additions, amino-terminal truncations and carboxy-terminal truncationssuch that these mutations encode MRNA which express a protein or proteinfragment of diagnostic, therapeutic or prophylactic use and would beuseful for screening for agonists and/or antagonists for DmLGICfunction.

A preferred aspect of this portion of the present invention is disclosedin FIGS. 1, 3, 5 and 6, which describes the four novel DNA moleculeswhich encode three forms of DmLGIC proteins.

The isolated nucleic acid molecules of the present invention may includea deoxyribonucleic acid molecule (DNA), such as genomic DNA andcomplementary DNA (cDNA), which may be single (coding or noncodingstrand) or double stranded, as well as synthetic DNA, such as asynthesized, single stranded polynucleotide. The isolated nucleic acidmolecule of the present invention may also include a ribonucleic acidmolecule (RNA).

The degeneracy of the genetic code is such that, for all but two aminoacids, more than a single codon encodes a particular amino acid. Thisallows for the construction of synthetic DNA that encodes the DmLGICprotein where the nucleotide sequence of the synthetic DNA differssignificantly from the nucleotide sequence of SEQ ID NOs: 1, 3, 5 and 6but still encodes the same DmLGIC protein as SEQ ID NO: 1, 3, 5 and 6.Such synthetic DNAs are intended to be within the scope of the presentinvention. If it is desired to express such synthetic DNAs in aparticular host cell or organism, the codon usage of such synthetic DNAscan be adjusted to reflect the codon usage of that particular host, thusleading to higher levels of expression of the DmLGIC protein in thehost. In other words, this redundancy in the various codons which codefor specific amino acids is within the scope of the present invention.Therefore, this invention is also directed to those DNA sequences whichencode RNA comprising alternative codons which code for the eventualtranslation of the identical amino acid, as shown below:

-   A=Ala=Alanine: codons GCA, GCC, GCG, GCU-   C=Cys=Cysteine: codons UGC, UGU-   D=Asp=Aspartic acid: codons GAC, GAU-   E=Glu=Glutamic acid: codons GAA, GAG-   F=Phe=Phenylalanine: codons UUC, UUU-   G=Gly=Glycine: codons GGA, GGC, GGG, GGU-   H=His=Histidine: codons CAC, CAU-   I=Ile=Isoleucine: codons AUA, AUC, AUU-   K=Lys=Lysine: codons AAA, AAG-   L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU-   M=Met=Methionine: codon AUG-   N=Asp=Asparagine: codons AAC, AAU-   P=Pro=Proline: codons CCA, CCC, CCG, CCU-   Q=Gln=Glutamine: codons CAA, CAG-   R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU-   S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU-   T=Thr=Threonine: codons ACA, ACC, ACG, ACU-   V=Val=Valine: codons GUA, GUC, GUG, GUU-   W=Trp=Tryptophan: codon UGG-   Y=Tyr=Tyrosine: codons UAC, UAU    Therefore, the present invention discloses codon redundancy which    may result in differing DNA molecules expressing an identical    protein. For purposes of this specification, a sequence bearing one    or more replaced codons will be defined as a degenerate variation.    Another source of sequence variation may occur through RNA editing,    as discussed infra. Such RNA editing may result in another form of    codon redundancy, wherein a change in the open reading frame does    not result in an altered amino acid residue in the expressed    protein. For whatever biological reason, a example can be found    within the present disclosure. The cDNA clones Ac15-4 and Ac15-25    encode a 485 amino acid protein as set forth in SEQ ID NO: 7, but 16    silent nucleotide changes occur when comparing the Ac15-25 open    reading frame sequence to the Ac15-4 open reading frame sequence.    Also included within the scope of this invention are mutations    either in the DNA sequence or the translated protein which do not    substantially alter the ultimate physical properties of the    expressed protein. For example, substitution of valine for leucine,    arginine for lysine, or asparagine for glutamine may not cause a    change in functionality of the polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate or a receptor for a ligand.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification. The nucleic acid molecules of the present inventionencoding a DmLGIC protein, in whole or in part, can be linked with otherDNA molecules, i.e, DNA molecules to which the DmLGIC coding sequenceare not naturally linked, to form “recombinant DNA molecules” whichencode a respective DmLGIC protein. The novel DNA sequences of thepresent invention can be inserted into vectors which comprise nucleicacids encoding DmLGIC or a functional equivalent. These vectors may becomprised of DNA or RNA; for most cloning purposes DNA vectors arepreferred. Typical vectors include plasmids, modified viruses,bacteriophage, cosmids, yeast artificial chromosomes, and other forms ofepisomal or integrated DNA that can encode a DmLGIC protein. It is wellwithin the purview of the skilled artisan to determine an appropriatevector for a particular gene transfer or other use.

Included in the present invention are DNA sequences that hybridize toSEQ ID NOs:1, 3, 5 and 6 under moderate to highly stringent conditions.By way of example, and not limitation, a procedure using conditions ofhigh stringency is as follows: Prehybridization of filters containingDNA is carried out for 2 hours to overnight at 65° C. in buffer composedof 6×SSC, 5× Denhardt's solution, and 100 μg/ml denatured salmon spermDNA. Filters are hybridized for 12 to 48 hrs at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5−20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 hr in a solution containing 2×SSC, 0.1% SDS. This is followedby a wash in 0.1×SSC, 0.1% SDS at 50° C. for 45 min. beforeautoradiography. Other procedures using conditions of high stringencywould include either a hybridization step carried out in 5×SSC, 5×Denhardt's solution, 50% formamide at 42° C. for 12 to 48 hours or awashing step carried out in 0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60minutes. Reagents mentioned in the foregoing procedures for carrying outhigh stringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.,:(Computational Molecular Biology, Lesk, A. M., ed. Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.. HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exists a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo and Lipton, 1988, SIAM J Applied Math48:1073). Methods commonly employed to determine identity or similaritybetween two sequences include, but are not limited to, those disclosedin Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, SanDiego, 1994, and Carillo and Lipton, 1988, SIAM J Applied Math 48:1073.Methods to determine identity and similarity are codified in computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, GCGprogram package (Devereux, et al, 1984, Nucleic Acids Research12(1):387), BLASTN, and FASTA (Altschul, et al., 1990, J Mol. Biol.215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO:1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations oralternative nucleotides per each 100 nucleotides of the referencenucleotide sequence of SEQ ID NO:1. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations or alternative nucleotide substitutions of the referencesequence may occur at the 5′ or 3′ terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence. One source of such a “mutation” or change which results in aless than 100% identity may occur through RNA editing. The process ofRNA editing results in modification of an MRNA molecule such that use ofthat modified mRNA as a template to generate a cloned cDNA may result inone or more nucleotide changes, which may or may not result in a codonchange. This RNA editing is known to be catalyzed by an RNA editase.Such an RNA editase is RNA adenosine deaminase, which converts anadenosine residue to an inosine residue, which tends to mimic a cytosineresidue. To this end, conversion of an mRNA residue from A to I willresult in A to G transitions in the coding and noncoding regions of acloned cDNA (e.g., see Hanrahan et al, 1999, Annals New York Acad. Sci.868:51-66; for a review see Bass, 1997, TIBS 22: 157-162). Similarly, bya polypeptide having an amino acid sequence having at least, forexample, 95% identity to a reference amino acid sequence of SEQ ID NO:2is intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of SEQ ID NO:2. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceof anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. Again, as noted above,RNA editing may result in a codon change which will result in anexpressed protein which differs in “identity” from other proteinsexpressed from “non-RNA edited” transcripts, which correspond directlyto the open reading frame of the genomic sequence.

The present invention also relates to a substantially purified form of arespective DmLGIC protein, which comprises the amino acid sequencedisclosed in FIG. 2, FIG. 4 and FIG. 6, and as set forth in SEQ IDNOs:2, 4 and 6, respectively. The disclosed DmLGIC proteins contain anopen reading frame of 426 (SEQ ID NO: 2), 422 amino acids (SEQ ID NO: 4)and 485 (SEQ ID NO:6) amino acids in length, as shown in FIGS. 2, 4 and6, and as follows: MQSPTSKLVE FRCLIALAIY LHALEQSIQH CHCVHGYRNNTESAELVSHY (SEQ ID NO:4) ESSLSLPDIL PIPSKTYDKN RAPKLLGQPT VVYFHVTVLSLDSINEESMT YVTDIFLAQS WRDPRLRLPE NMSEQYRILD VDWLHSIWRP DCFFKNAKKVTFHEMSIPNH YLWLYHDKTL LYMSKLTLVL SCAMKFESYP HDTQICSMMI ESLSHTVEDLVFIWNMTDPL VVNTEIELPQ LDISNNYTTD CTIEYSTGNF TCLAIVFNLR RRLGYHLFHTYIPSALIVVM SWISFWIKPE AIPARVTLGV TSLLTLATQN TQSQQSLPPV SPVKAIDVWMSSCSVFVFLS LMEFAVVNNF MGPVATKAMK GYSDENISDL DDLKSALQHH RESIIEPQYDTFCHGHATAI YIDKFSRFFF PFSFFILNIV YWTTFL*; MQSPTSKLVE FRCLIALAIYLHALEQSIQH CHCVHGYRNN TESAELVSHY (SEQ ID NO:4) ESSLSLPDIL PIPSKTYDKNRAPKLLGQPT VVYFHVTVLS LDSINEESMT YVTDIFLAQS WRDPRLRLPE NMSEQYRILDVDWLHSIWRP DCFFKNAKKV TFHEMSIPNH YLWLYHDKTL LYMSKLTLVL SCAMKFESYPHDTQICSMMI ESLSHTVEDL VFIWNMTDPL VVNTEIELPQ LDISNNYTTD CTIEYSTGNFTCLAIVFNLR RRLGYHLFHT YIPSALIVVM SWISFWIKPE AIPARVTLGV TSLLTLATQNTQSQQSLPPV SYVKAIDVWM SSCSVFVFLS LMEFAVVNNF MGPVATKAMK GYSDENISDLDDLKHHRESI IEPQYDTFCH GHATAIYIDK FSRFFFPFSF FILNIVYWTT FL*; and,MVFQIIILVI CTICMKHYAK GEFQQSLAIT DILPEDIKRY DKMRPPKKEG (SEQ ID NO:7)QPTIVYFHVT VMGLDSIDEN SMTYVADVFF AQTWKDHRLR LPENMTQEYR LLEVDWLKNMWRPDSFFKNA KSVTFQTMTI PNHYMWLYKD KTILYMVKLT LKLCCIMNFA IYPHDTQECKLQMESLSHTT DDLIFQWDPT TPLVVDENIE LPQVALIRNE TADCTQVYST GNFTCLEVVFTLKRRLVYYV FNTYIPTCMI VIMSWVSFWI KPEAAPARVT LGVTSLLTLS TQHAKSQSSLPPVSYLKAVD AFMSVCTVFV FMALMEYCLI NIVLSDTPIP KPMAYPPKPV AGDGPKKEGEGAPPGGSNST ASKQQATMLP LADEKIEKIE KIFDEMTKNR RIVTTTRRVV RPPLDADGPWIPRQESRIIL TPTIAPPPPP PQPAAPEPEL PKPKLTPAQE RLKRAIYIDR SSRVLFPALFASLNGIYWCV FYEYL*.

The present invention also relates to biologically active fragmentsand/or mutants of the DmLGIC protein comprising the amino acid sequenceas set forth in SEQ ID NOs:2, 4, and 7, including but not necessarilylimited to amino acid substitutions, deletions, additions, aminoterminal truncations and carboxy-terminal truncations such that thesemutations provide for proteins or protein fragments of diagnostic,therapeutic or prophylactic use and would be useful for screening foragonists and/or antagonists of DmLGIC function.

Another preferred aspect of the present invention relates to asubstantially purified, fully processed LGIC protein obtained from arecombinant host cell containing a DNA expression vector comprises anucleotide sequence as set forth in SEQ ID NOs: 1, 3, 5, and/or 6 andexpresses the respective DmLGIC precursor protein. It is especiallypreferred that the recombinant host cell be a eukaryotic host cell, suchas a mammalian cell line, or Xenopus oocytes, as noted above.

As with many proteins, it is possible to modify many of the amino acidsof DmLGIC protein and still retain substantially the same biologicalactivity as the wild type protein. Thus this invention includes modifiedDmLGIC polypeptides which have amino acid deletions, additions, orsubstitutions but that still retain substantially the same biologicalactivity as a respective, corresponding DmLGIC. It is generally acceptedthat single amino acid substitutions do not usually alter the biologicalactivity of a protein (see, e.g., Molecular Biology of the Gene, Watsonet al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc.,page 226; and Cunningham & Wells, 1989, Science 244:1081-1085).Accordingly, the present invention includes polypeptides where one aminoacid substitution has been made in SEQ ID NO:2, 4, and/or 7 wherein thepolypeptides still retain substantially the same biological activity asa corresponding DmLGIC protein. The present invention also includespolypeptides where two or more amino acid substitutions have been madein SEQ ID NO:2, 4, or 7 wherein the polypeptides still retainsubstantially the same biological activity as a corresponding DmLGICprotein. In particular, the present invention includes embodiments wherethe above-described substitutions are conservative substitutions.

One skilled in the art would also recognize that polypeptides that arefunctional equivalents of DmLGIC and have changes from the DmLGIC aminoacid sequence that are small deletions or insertions of amino acidscould also be produced by following the same guidelines, (i.e,minimizing the differences in amino acid sequence between DmLGIC andrelated proteins. Small deletions or insertions are generally in therange of about 1 to 5 amino acids. The effect of such small deletions orinsertions on the biological activity of the modified DmLGIC polypeptidecan easily be assayed by producing the polypeptide synthetically or bymaking the required changes in DNA encoding DmLGIC and then expressingthe DNA recombinantly and assaying the protein produced by suchrecombinant expression.

The present invention also includes truncated forms of DmLGIC whichcontain the region comprising the active site of the enzyme. Suchtruncated proteins are useful in various assays described herein, forcrystallization studies, and for structure-activity-relationshipstudies.

The present invention also relates to membrane-containing crude lysatesor substantially purified subcellular membrane fractions from therecombinant host cells (both prokaryotic and eukaryotic as well as bothstably and transiently transformed cells) which contain the nucleic acidmolecules of the present invention. These recombinant host cells expressDmLGIC or a functional equivalent, which becomes post translationallyassociated with the cell membrane in a biologically active fashion.These subcellular membrane fractions will comprise either wild-type ormutant forms of DmLGIC at levels substantially above endogenous levelsand hence will be useful in assays to select modulators of DmLGICproteins or channels. In other words, a specific use for suchsubcellular membranes involves expression of DmLGIC within therecombinant cell followed by isolation and substantial purification ofthe membranes away from other cellular components and subsequent use inassays to select for modulators, such as agonist or antagonists of theprotein or biologically active channel comprising one or more of theproteins disclosed herein. Alternatively, the lysed cells, containingthe membranes, may be used directly in assays to select for modulatorsof the recombinantly expressed protein(s) disclosed herein. Therefore,another preferred aspect of the present invention relates to asubstantially purified membrane preparation or lysed recombinant cellcomponents which include membranes, which has been obtained from arecombinant host cell transformed or transfected with a DNA expressionvector which comprises and appropriately expresses a complete openreading frame as set forth in SEQ ID NOs: 1, 3, 5, and/or 6, resultingin a functional, processed form of the respective single, homomultimeror heteromultimer DmLGIC receptor. It is especially preferred is thatthe recombinant host cell be a eukaryotic host cell, such as a mammaliancell line, or Xenopus oocytes, as noted above.

To this end, a preferred aspect of the present invention is a functionalDmLGIC channel receptor, comprised of either a single channel protein ora channel comprising multiple subunits, referred to herein as ahomomultimer channel or a heteromultimer channel. Therefore, a singlechannel may be comprised of a protein as disclosed in SEQ ID NOs: 2, 4or 7, or a biologically active equivalent thereof (i.e., a alteredchannel protein which still functions in a similar fashion to that of awild-type channel receptor). A homomultimer channel receptor complexwill comprise more than one polypeptide selected from the disclosedgroup of SEQ ID NOs: 2, 4 and 7, as well as biologically activeequivalents. A heteromultimer channel receptor complex will comprisemultiple subunits wherein at least 2 of the 3 proteins disclosed hereincontribute to channel formation, or where at least one of the proteinsassociates with additional proteins or channel components to provide foran active channel receptor complex. Therefore, the present inventionadditionally relates to substantially purified channels as describedherein, as well as substantially purified membrane preparations,partially purified membrane preparations, or cell lysates which containthe functional single, homomultimer or heteromultimer channels describedherein.

The present invention also relates to isolated nucleic acid moleculeswhich are fusion constructions expressing fusion proteins useful inassays to identify compounds which modulate wild-type DmLGIC activity,as well as generating antibodies against DmLGIC. One aspect of thisportion of the invention includes, but is not limited to, glutathioneS-transferase (GST)-DmLGIC fusion constructs. Recombinant GST-DmLGICfusion proteins may be expressed in various expression systems,including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using abaculovirus expression vector (pAcG2T, Pharmingen). Another aspectinvolves DmLGIC fusion constructs linked to various markers, includingbut not limited to GFP (Green fluorescent protein), the MYC epitope, andGST. Again, any such fusion constructs may be expressed in the cell lineof interest and used to screen for modulators of one or more of theDmLGIC proteins disclosed herein.

A preferred aspect for screening for modulators of DmLGIC activity is anexpression system for the electrophysiological-based assays formeasuring ligand-gated ion channel activity comprising injecting the DNAmolecules of the present invention into Xenopus laevis oocytes. Thegeneral use of Xenopus oocytes in the study of ion channel activity isknown in the art (Dascal, 1987, Crit. Rev. Biochem. 22: 317-317; Lester,1988, Science 241: 1057-1063; see also Methods of Enzymology, Vol. 207,1992, Ch. 14-25, Rudy and Iverson, ed., Academic Press, Inc., New York).An improved method exists for measuring channel activity and modulationby agonists and/or antagonists which is several-fold more sensitive thanprevious techniques. The Xenopus oocytes are injected with nucleic acidmaterial, including but not limited to DNA, mRNA or cRNA which encode agated-channel, wherein channel activity may be measured as well asresponse of the channel to various modulators. Ion channel activity ismeasured by utilizing a holding potential more positive than thereversal potential for chloride (i.e, greater than −30 mV), preferablyabout 0 mV. This alteration in assay measurement conditions hasresulting in a 10-fold increase in sensitivity of the assay tomodulation by ivermectin phosphate. Therefore, this improved assayallows screening and selecting for compounds which modulate LGICactivity at levels which were previously thought to be undetectable.

Any of a variety of procedures may be used to clone DmLGIC. Thesemethods include, but are not limited to, (1) a RACE PCR cloningtechnique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85:8998-9002). 5′ and/or 3′ RACE may be performed to generate a full-lengthcDNA sequence. This strategy involves using gene-specificoligonucleotide primers for PCR amplification of DmLGIC cDNA. Thesegene-specific primers are designed through identification of anexpressed sequence tag (EST) nucleotide sequence which has beenidentified by searching any number of publicly available nucleic acidand protein databases; (2) direct functional expression of the DmLGICcDNA following the construction of a DmLGIC-containing cDNA library inan appropriate expression vector system; (3) screening aDmLGIC-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a labeled degenerate oligonucleotide probe designedfrom the amino acid sequence of the DmLGIC protein; (4) screening aDmLGIC-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a partial cDNA encoding the DmLGIC protein. Thispartial cDNA is obtained by the specific PCR amplification of DmLGIC DNAfragments through the design of degenerate oligonucleotide primers fromthe amino acid sequence known for other kinases which are related to theDmLGIC protein; (5) screening a DmLGIC-containing cDNA libraryconstructed in a bacteriophage or plasmid shuttle vector with a partialcDNA or oligonucleotide with homology to a mammalian DmLGIC protein.This strategy may also involve using gene-specific oligonucleotideprimers for PCR amplification of DmLGIC cDNA identified as an EST asdescribed above; or (6) designing 5′ and 3′ gene specificoligonucleotides using SEQ ID NO: 1, 3, and 5 as a template so thateither the full-length cDNA may be generated by known RACE techniques,or a portion of the coding region may be generated by these same knownRACE techniques to generate and isolate a portion of the coding regionto use as a probe to screen one of numerous types of cDNA and/or genomiclibraries in order to isolate a full-length version of the nucleotidesequence encoding DmLGIC. Alternatively, the DmLGIC cDNA may be clonedas described in Example Section 1. Briefly, partial sequencespotentially encoding two novel ligand gated ion channel genes, AC05 andAC15, were identified in the Drosophila genome sequencing project usingthe Extended Smith Waterman algorithm. The query sequence was the C.elegans glutamate gated ion channel avr-15a peptide sequence (accessionnumber-AJ000538), and the DNA database searched was publicly availableDrosophila high throughput genomic sequences. The search was performedon a Compugen Biocel XLP hardware search engine (Petach Tikva, Israel).Both sequences entered into the database contained predicted introns.Primers specific to either Ac05 or Ac15 were designed based on thedatabase sequences. With these primer combinations, RT-PCR on whole flytotal RNA followed by TA cloning was performed for both genes. Fragmentsof approximately 500 bp in length for both Ac05 and Ac15 were isolatedand verified by DNA sequencing. PolyA⁺ RNA was purified from whole bodyOregon R Drosophila and used to generate the double-stranded cDNA. 5′and 3′ RACE fragments were obtained for both genes by 1^(st) round PCRand nested PCR. The resulting fragment sizes were ˜1.3 kb for Ac05 and˜1.8 kb for Ac15 in 3′-RACE. In 5′-RACE Ac05 and Ac15 both have fragmentsizes of ˜1 kb. The PCR products were cloned into a pCR2.1-TOPO vector.Miniprep DNA samples were screened by restriction digestion to separatespliced from unspliced clones. Using the sequences obtained from the 5′and 3′ RACE products, PCR primers for both genes were designed togenerate full-length clones. cDNA clones Ac05-10 and Ac05-11 weregenerated using primers Ac05 F1 and R1 for 1^(st) round PCR and primersAc05 F1 and R2 for 2^(nd) round PCR. cDNA clones Ac15-4 and Ac15-25 weregenerated using primers Ac15 F1 and R1 for 1^(st) round PCR. The PCRproducts were cloned into pCR2.1-TOPO vector. Two clones of Ac05 wereidentified: Ac05-10 (1518 bp) and Ac05-11.(1506 bp). The clones areidentical but for a 4 amino acid insertion within the M3-M4intracellular loop in Ac05-10 (a Ser-Ala-Leu-Gln insertion from aminoacid residue 375 through amino acid residue 378, as set forth in SEQ IDNO:2). Two clones of Ac15 were identified: Ac15-4 (2073 bp) and Ac15-25(2034 bp), which predict the same protein sequence but differ in 16nucleotides within 1455 nucleotide open reading frame.

It is readily apparent to those skilled in the art that other types oflibraries, as well as libraries constructed from other cell types-orspecies types, may be useful for isolating a DmLGIC-encoding DNA or aDmLGIC homologue. Other types of libraries include, but are not limitedto, cDNA libraries derived from other cells.

It is readily apparent to those skilled in the art that suitable cDNAlibraries may be prepared from cells or cell lines which have DmLGICactivity. The selection of cells or cell lines for use in preparing acDNA library to isolate a cDNA encoding DmLGIC may be done by firstmeasuring cell-associated DmLGIC activity using any known assayavailable for such a purpose.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Complementary DNA libraries may also be obtained from numerouscommercial sources, including but not limited to Clontech Laboratories,Inc. and Stratagene.

It is also readily apparent to those skilled in the art that DNAencoding DmLGIC may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in Sambrook, et al., supra.One may prepare genomic libraries, especially in P1 artificialchromosome vectors, from which genomic clones containing the DmLGIC canbe isolated, using probes based upon the DmLGIC nucleotide sequencesdisclosed herein. Methods of preparing such libraries are known in theart (Ioannou et al., 1994, Nature Genet. 6:84-89).

In order to clone a DmLGIC gene by one of the preferred methods, theamino acid sequence or DNA sequence of a DmLGIC or a homologous proteinmay be necessary. To accomplish this, a respective DmLGIC protein may bepurified and the partial amino acid sequence determined by automatedsequenators. It is not necessary to determine the entire amino acidsequence, but the linear sequence of two regions of 6 to 8 amino acidscan be determined for the PCR amplification of a partial DmLGIC DNAfragment. Once suitable amino acid sequences have been identified, theDNA sequences capable of encoding them are synthesized. Because thegenetic code is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the DmLGIC sequence but others in theset will be capable of hybridizing to DmLGIC DNA even in the presence ofDNA oligonucleotides with mismatches. The mismatched DNAoligonucleotides may still sufficiently hybridize to the DmLGIC DNA topermit identification and isolation of DmLGIC encoding DNA.Alternatively, the nucleotide sequence of a region of an expressedsequence may be identified by searching one or more available genomicdatabases. Gene-specific primers may be used to perform PCRamplification of a cDNA of interest from either a cDNA library or apopulation of cDNAs. As noted above, the appropriate nucleotide sequencefor use in a PCR-based method may be obtained from SEQ ID NO: 1, 3, 5 or6 either for the purpose of isolating overlapping 5′ and 3′ RACEproducts for generation of a full-length sequence coding for DmLGIC, orto isolate a portion of the nucleotide sequence coding for DmLGIC foruse as a probe to screen one or more cDNA- or genomic-based libraries toisolate a full-length sequence encoding DmLGIC or DmLGIC-like proteins.

This invention also includes vectors containing a DmLGIC gene, hostcells containing the vectors, and methods of making substantially pureDmLGIC protein comprising the steps of introducing the DmLGIC gene intoa host cell, and cultivating the host cell under appropriate conditionssuch that DmLGIC is produced. The DmLGIC so produced may be harvestedfrom the host cells in conventional ways. Therefore, the presentinvention also relates to methods of expressing the DmLGIC protein andbiological equivalents disclosed herein, assays employing these geneproducts, recombinant host cells which comprise DNA constructs whichexpress these proteins, and compounds identified through these assayswhich act as agonists or antagonists of DmLGIC activity.

The cloned DmLGIC cDNA obtained through the methods described above maybe recombinantly expressed by molecular cloning into an expressionvector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pCR2.1-TOPO, pBlueBacHis2or pLITMUS28, as well as other examples, listed infra) containing asuitable promoter and other appropriate transcription regulatoryelements, and transferred into prokaryotic or eukaryotic host cells toproduce recombinant DmLGIC. Expression vectors are defined herein as DNAsequences that are required for the transcription of cloned DNA and thetranslation of their mRNAs in an appropriate host. Such vectors can beused to express eukaryotic DNA in a variety of hosts such as bacteria,blue green algae, plant cells, insect cells and animal cells.Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. To determine the DmLGIC cDNA sequence(s) that yields optimallevels of DmLGIC, cDNA molecules including but not limited to thefollowing can be constructed: a cDNA fragment containing the full-lengthopen reading frame for DmLGIC as well as various constructs containingportions of the cDNA encoding only specific domains of the protein orrearranged domains of the protein. All constructs can be designed tocontain none, all or portions of the 5′ and/or 3′ untranslated region ofa DmLGIC cDNA. The expression levels and activity of DmLGIC can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the DmLGIC cDNA cassette yielding optimal expression in transientassays, this DmLGIC cDNA construct is transferred to a variety ofexpression vectors (including recombinant viruses), including but notlimited to those for mammalian cells, plant cells, insect cells,oocytes, bacteria, and yeast cells. Techniques for such manipulationscan be found described in Sambrook, et al., supra, are well known andavailable to the artisan of ordinary skill in the art. Therefore,another aspect of the present invention includes host cells that havebeen engineered to contain and/or express DNA sequences encoding theDmLGIC. An expression vector containing DNA encoding a DmLGIC-likeprotein may be used for expression of DmLGIC in a recombinant host cell.Such recombinant host cells can be cultured under suitable conditions toproduce DmLGIC or a biologically equivalent form. Expression vectors mayinclude, but are not limited to, cloning vectors, modified cloningvectors, specifically designed plasmids or viruses. Commerciallyavailable mammalian expression vectors which may be suitable forrecombinant DmLGIC expression, include but are not limited to,pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega),pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs),pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMC1neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), and IZD35 (ATCC 37565). Also, a variety ofbacterial expression vectors may be used to express recombinant DmLGICin bacterial cells. Commercially available bacterial expression vectorswhich may be suitable for recombinant DmLGIC expression include, but arenot limited to pCR2.1 (Invitrogen), pET11a (Novagen), lambda gt11(Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungalcell expression vectors may be used to express recombinant DmLGIC infungal cells. Commercially available fungal cell expression vectorswhich may be suitable for recombinant DmLGIC expression include but arenot limited to pYES2 (Invitrogen) and Pichia expression vector(Invitrogen). Also, a variety of insect cell expression vectors may beused to express recombinant protein in insect cells. Commerciallyavailable insect cell expression vectors which may be suitable forrecombinant expression of DmLGIC include but are not limited topBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to, bacteria such as E. coli, fungal cells such as yeast,mammalian cells including, but not limited to, cell lines of bovine,porcine, monkey and rodent origin; and insect cells including but notlimited to Drosophila and silkworm derived cell lines. For instance, oneinsect expression system utilizes Spodoptera frugiperda (Sf21) insectcells (Invitrogen) in tandem with a baculovirus expression vector(pAcG2T, Pharmingen). Also, mammalian species which may be suitable andwhich are commercially available, include but are not limited to, Lcells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCCHTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70),COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3(ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCCCRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL209).

The specificity of binding of compounds showing affinity for DmLGIC isshown by measuring the affinity of the compounds for recombinant cellsexpressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto DmLGIC or that inhibit the binding of a known, radiolabeled ligand ofDmLGIC to these cells, or membranes prepared from these cells, providesan effective method for the rapid selection of compounds with highaffinity for DmLGIC. Such ligands need not necessarily be radiolabeledbut can also be nonisotopic compounds that can be used to displace boundradiolabeled compounds or that can be used as activators in functionalassays. Compounds identified by the above method are likely to beagonists or antagonists of DmLGIC and may be peptides, proteins, ornon-proteinaceous organic molecules.

Accordingly, the present invention is directed to methods for screeningfor compounds which modulate the expression of DNA or RNA encoding aDmLGIC protein as well as compounds which effect the function of theDmLGIC protein. Methods for identifying agonists and antagonists ofother receptors are well known in the art and can be adapted to identifyagonists and antagonists of a DmLGIC channel. For example, Cascieri etal. (1992, Molec. Pharmacol. 41:1096-1099) describe a method foridentifying substances that inhibit agonist binding to rat neurokininreceptors and thus are potential agonists or antagonists of neurokininreceptors. The method involves transfecting COS cells with expressionvectors containing rat neurokinin receptors, allowing the transfectedcells to grow for a time sufficient to allow the neurokinin receptors tobe expressed, harvesting the transfected cells and resuspending thecells in assay buffer containing a known radioactively labeled agonistof the neurokinin receptors either in the presence or the absence of thesubstance, and then measuring the binding of the radioactively labeledknown agonist of the neurokinin receptor to the neurokinin receptor. Ifthe amount of binding of the known agonist is less in the presence ofthe substance than in the absence of the substance, then the substanceis a potential agonist or antagonist of the neurokinin receptor. Wherebinding of the substance such as an agonist or antagonist to DmLGIC ismeasured, such binding can be measured by employing a labeled substanceor agonist. The substance or agonist can be labeled in any convenientmanner known to the art, e.g., radioactively, fluorescently,enzymatically.

As noted above in regard to the use of Xenopus oocytes to express aDmLGIC gene of interest, the present invention is directed to methodsfor screening for compounds which modulate the expression of DNA or RNAencoding a DmLGIC protein. Compounds which modulate these activities maybe DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.Compounds may modulate by increasing or attenuating the expression ofDNA or RNA encoding DmLGIC, or the function of the DmLGIC-basedchannels. Compounds that modulate the expression of DNA or RNA encodingDmLGIC or the biological function (i.e., channel activation by histamineor other ligands and/or compounds which activate the wild type channel)thereof may be detected by a variety of assays. The assay may be asimple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function (i.e., effect of channel activity) of a testsample with the levels of expression or function in a standard sample.Kits containing DmLGIC, antibodies to DmLGIC, or modified DmLGIC may beprepared by known methods for such uses.

To this end, the present invention relates in part to methods ofidentifying a substance which modulates DmLGIC receptor activity, whichinvolves:

(a) combining a test substance in the presence and absence of a DmLGICreceptor protein wherein said DmLGIC receptor protein comprises theamino acid sequence as set forth in SEQ ID NO:, 4, and/or 7; and,

(b) measuring and comparing the effect of the test substance in thepresence and absence of the DmLGIC receptor protein.

In addition, several specific embodiments are disclosed herein to showthe diverse type of screening or selection assay which the skilledartisan may utilize in tandem with an expression vector directing theexpression of the DmLGIC receptor protein. Methods for identifyingagonists and antagonists of other receptors are well known in the artand can be adapted to identify agonists and antagonists of DmLGIC.Therefore, these embodiments are presented as examples and not aslimitations. To this end, the present invention includes assays by whichDmLGIC modulators (such as agonists and antagonists) may be identified.Accordingly, the present invention includes a method for determiningwhether a substance is a potential agonist or antagonist of DmLGIC thatcomprises:

(a) transfecting or transforming cells with an expression vector thatdirects expression of DmLGIC in the cells, resulting in test cells;

(b) allowing the test cells to grow for a time sufficient to allowDmLGIC to be expressed and for a functional channel to be generated;

(c) exposing the cells to a labeled ligand of DmLGIC in the presence andin the absence of the substance;

(d) measuring the binding of the labeled ligand to the DmLGIC channel;where if the amount of binding of the labeled ligand is less in thepresence of the substance than in the absence of the substance, then thesubstance is a potential agonist or antagonist of DmLGIC.

The conditions under which step (c) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells may be harvested and resuspended in the presence of thesubstance and the labeled ligand. In a modification of theabove-described method, step (c) is modified in that the cells are notharvested and resuspended but rather the radioactively labeled knownagonist and the substance are contacted with the cells while the cellsare attached to a substratum, e.g., tissue culture plates.

The present invention also includes a method for determining whether asubstance is capable of binding to DmLGIC, i.e., whether the substanceis a potential agonist or an antagonist of DmLGIC channel activation,where the method comprises:

(a) transfecting or transforming cells with an expression vector thatdirects the expression of DmLGIC in the cells, resulting in test cells;

(b) exposing the test cells to the substance;

(c) measuring the amount of binding of the substance to DmLGIC;

(d) comparing the amount of binding of the substance to DmLGIC in thetest cells with the amount of binding of the substance to control cellsthat have not been transfected with DmLGIC;

wherein if the amount of binding of the substance is greater in the testcells as compared to the control cells, the substance is capable ofbinding to DmLGIC. Determining whether the substance is actually anagonist or antagonist can then be accomplished by the use of functionalassays such as, e.g., the assay involving the use of promiscuousG-proteins described below.

The conditions under which step (b) of the method is practiced areconditions that are typically used in the art for the study ofprotein-ligand interactions: e.g., physiological pH; salt conditionssuch as those represented by such commonly used buffers as PBS or intissue culture media; a temperature of about 4° C. to about 55° C. Thetest cells are harvested and resuspended in the presence of thesubstance.

The above described assays may be functional assays, whereelectrophysiological assays (e.g., see Example 2) may be carried out intransfected mammalian cell lines as well as Xenopus oocytes to measurethe various effects test compounds may have on the ability of a knownligand (such as histamine or glutamate) to activate the channel, or fora test compound to modulate activity in and of itself (similar to theeffect of ivermectin on known GluCl channels). Therefore, the skilledartisan will be comfortable adapting the cDNA clones of the presentinvention to known methodology to both initially and secondary screensto select for compounds that bind and/or activate the functional DmLGICchannels of the present invention.

The DNA molecules, RNA molecules, recombinant protein and antibodies ofthe present invention may be used to screen and measure levels ofDmLGIC. The recombinant proteins, DNA molecules, RNA molecules andantibodies lend themselves to the formulation of kits suitable for thedetection and typing of DmLGIC. Such a kit would comprise acompartmentalized carrier suitable to hold in close confinement at leastone container. The carrier would further comprise reagents such asrecombinant DmLGIC or anti-DmLGIC antibodies suitable for detectingDmLGIC. The carrier may also contain a means for detection such aslabeled antigen or enzyme substrates or the like.

The assays described above can be carried out with cells that have beentransiently or stably transfected with DmLGIC. The expression vector maybe introduced into host cells via any one of a number of techniquesincluding but not limited to transformation, transfection, protoplastfusion, and electroporation. Transfection is meant to include any methodknown in the art for introducing DmLGIC into the test cells. Forexample, transfection includes calcium phosphate or calcium chloridemediated transfection, lipofection, infection with a retroviralconstruct containing DmLGIC, and electroporation. The expressionvector-containing cells are individually analyzed to determine whetherthey produce DmLGIC protein. Identification of DmLGIC expressing cellsmay be done by several means, including but not limited to immunologicalreactivity with anti-DmLGIC antibodies, labeled ligand binding, and/orthe presence of host cell-associated DmLGIC activity.

The specificity of binding of compounds showing affinity for DmLGIC isshown by measuring the affinity of the compounds for recombinant cellsexpressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto DmLGIC or that inhibit the binding of a known, radiolabeled ligand ofDmLGIC to these cells, or membranes prepared from these cells, providesan effective method for the rapid selection of compounds with highaffinity for DmLGIC. Such ligands need not necessarily be radiolabeledbut can also be nonisotopic compounds that can be used to displace boundradiolabeled compounds or that can be used as activators in functionalassays. Compounds identified by the above method are likely to beagonists or antagonists of DmLGIC and may be peptides, proteins, ornon-proteinaceous organic molecules.

Accordingly, the present invention is directed to methods for screeningfor compounds which modulate the expression of DNA or RNA encoding aDmLGIC protein as well as compounds which effect the function of theDmLGIC protein. Methods for identifying agonists and antagonists ofother receptors are well known in the art and can be adapted to identifyagonists and antagonists of DmLGIC. For example, Cascieri et al. (1992,Molec. Pharnacol. 41:1096-1099) describe a method for identifyingsubstances that inhibit agonist binding to rat neurokinin receptors andthus are potential agonists or antagonists of neurokinin receptors. Themethod involves transfecting COS cells with expression vectorscontaining rat neurokinin receptors, allowing the transfected cells togrow for a time sufficient to allow the neurokinin receptors to beexpressed, harvesting the transfected cells and resuspending the cellsin assay buffer containing a known radioactively labeled agonist of theneurokinin receptors either in the presence or the absence of thesubstance, and then measuring the binding of the radioactively labeledknown agonist of the neurokinin receptor to the neurokinin receptor. Ifthe amount of binding of the known agonist is less in the presence ofthe substance than in the absence of the substance, then the substanceis a potential agonist or antagonist of the neurokinin receptor. Wherebinding of the substance such as an agonist or antagonist to ismeasured, such binding can be measured by employing a labeled substanceor agonist. The substance or agonist can be labeled in any convenientmanner known to the art, e.g., radioactively, fluorescently,enzymatically.

Therefore, the specificity of binding of compounds having affinity forDmLGIC shown by measuring the affinity of the compounds for recombinantcells expressing the cloned receptor or for membranes from these cells.Expression of the cloned receptor and screening for compounds that bindto DmLGIC or that inhibit the binding of a known, radiolabeled ligand ofDmLGIC (such as glutamate, ivermectin or nodulasporic acid) to thesecells, or membranes prepared from these cells, provides an effectivemethod for the rapid selection of compounds with high affinity forDmLGIC. Such ligands need not necessarily be radiolabeled but can alsobe nonisotopic compounds that can be used to displace bound radiolabeledcompounds or that can be used as activators in functional assays.Compounds identified by the above method again are likely to be agonistsor antagonists of DmLGIC and may be peptides, proteins, ornon-proteinaceous organic molecules. As noted elsewhere in thisspecification, compounds may modulate by increasing or attenuating theexpression of DNA or RNA encoding DmLGIC, or by acting as an agonist orantagonist of the DmLGIC receptor protein. Again, these compounds thatmodulate the expression of DNA or RNA encoding DmLGIC or the biologicalfunction thereof may be detected by a variety of assays. The assay maybe a simple “yes/no” assay to determine whether there is a change inexpression or function. The assay may be made quantitative by comparingthe expression or function of a test sample with the levels ofexpression or function in a standard sample.

Expression of DmLGIC DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

Following expression of DmLGIC in a host cell, DmLGIC protein may berecovered to provide DmLGIC protein in active form. Several DmLGICprotein purification procedures are available and suitable for use.Recombinant DmLGIC protein may be purified from cell lysates andextracts by various combinations of, or individual application of saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. In addition, recombinant DmLGICprotein can be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length DmLGIC protein, or polypeptide fragments ofDmLGIC protein.

Polyclonal or monoclonal antibodies may be raised against DmLGIC or asynthetic peptide (usually from about 9 to about 25 amino acids inlength) from a portion of DmLGIC as disclosed in SEQ ID NOs:2, 4, and/or7. Monospecific antibodies to DmLGIC are purified from mammalianantisera containing antibodies reactive against DmLGIC or are preparedas monoclonal antibodies reactive with DmLGIC using the technique ofKohler and Milstein (1975, Nature 256: 495497). Monospecific antibody asused herein is defined as a single antibody species or multiple antibodyspecies with homogenous binding characteristics for DmLGIC. Homogenousbinding as used herein refers to the ability of the antibody species tobind to a specific antigen or epitope, such as those associated withDmLGIC, as described above. Human DmLGIC-specific antibodies are raisedby immunizing animals such as mice, rats, guinea pigs, rabbits, goats,horses and the like, with an appropriate concentration of DmLGIC proteinor a synthetic peptide generated from a portion of DmLGIC with orwithout an immune adjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 mg and about 1000 mg of DmLGIC proteinassociated with an acceptable immune adjuvant. Such acceptable adjuvantsinclude, but are not limited to, Freund's complete, Freund's incomplete,alum-precipitate, water in oil emulsion containing Cornyebacteriumparvum and tRNA. The initial immunization consists of DmLGIC protein orpeptide fragment thereof in, preferably, Freund's complete adjuvant atmultiple sites either subcutaneously (SC), intraperitoneally (IP) orboth. Each animal is bled at regular intervals, preferably weekly,. todetermine antibody titer. The animals may or may not receive boosterinjections following the initial immunization. Those animals receivingbooster injections are generally given an equal amount of DmLGIC inFreund's incomplete adjuvant by the same route. Booster injections aregiven at about three week intervals until maximal titers are obtained.At about 7 days after each booster immunization or about weekly after asingle immunization, the animals are bled, the serum collected, andaliquots are stored at about −20° C.

Monoclonal antibodies (mAb) reactive with DmLGIC are prepared byimmunizing inbred mice, preferably Balb/c, with DmLGIC protein. The miceare immunized by the IP or SC route with about 1 mg to about 100 mg,preferably about 10 mg, of DmLGIC protein in about 0.5 ml buffer orsaline incorporated in an equal volume of an acceptable adjuvant, asdiscussed above. Freund's complete adjuvant is preferred. The micereceive an initial immunization on day 0 and are rested for about 3 toabout 30 weeks. Immunized mice are given one or more boosterimmunizations of about 1 to about 100 mg of DmLGIC in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin supplementedDulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.Supernatant fluids are collected form growth positive wells on aboutdays 14, 18, and 21 and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using DmLGIC asthe antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the mAb. Hybridoma cellsfrom antibody positive wells are cloned by a technique such as the softagar technique of MacPherson, 1973, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-DmLGIC mAb is carried out by growing thehybridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of DmLGIC inbody fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for DmLGIC peptide fragments, or arespective full-length DmLGIC.

DmLGIC antibody affinity columns are made, for example, by adding theantibodies to Affigel-10 (Biorad), a gel support which is pre-activatedwith N-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing full-length DmLGIC orDmLGIC protein fragments are slowly passed through the column. Thecolumn is then washed with phosphate buffered saline until the opticaldensity (A280) falls to background, then the protein is eluted with 0.23M glycine-HCl (pH 2.6). The purified DmLGIC protein is then dialyzedagainst phosphate buffered saline.

The present invention also relates to a non-human transgenic animalwhich is useful for studying the ability of a variety of compounds toact as modulators of DmLGIC, or any alternative functional DmLGIC invivo by providing cells for culture, in vitro. In reference to thetransgenic animals of this invention, reference is made to transgenesand genes. As used herein, a transgene is a genetic construct includinga gene. The transgene is integrated into one or more chromosomes in thecells in an animal by methods known in the art. Once integrated, thetransgene is carried in at least one place in the chromosomes of atransgenic animal. Of course, a gene is a nucleotide sequence thatencodes a protein, such as one or a combination of the cDNA clonesdescribed herein. The gene and/or transgene may also include geneticregulatory elements and/or structural elements known in the art. A typeof target cell for transgene introduction is the embryonic stem cell(ES). ES cells can be obtained from pre-implantation embryos cultured invitro and fused with embryos (Evans et al., 1981, Nature 292:154-156;Bradley et al., 1984, Nature 309:255-258; Gossler et al., 1986, Proc.Natl. Acad. Sci. USA 83:9065-9069; and Robertson et al., 1986 Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby a variety of standard techniques such as DNA transfection,microinjection, or by retrovirus-mediated transduction. The resultanttransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The introduced ES cells thereafter colonize the embryoand contribute to the germ line of the resulting chimeric animal(Jaenisch, 1988, Science 240: 1468-1474). It will also be within thepurview of the skilled artisan to produce transgenic or knock-outinvertebrate animals (e.g., C. elegans) which express the DmLGICtransgene in a wild type C. elegans LGIC background as well in C.elegans mutants knocked out for one or both of the C. elegans LGICsubunits.

Pharmaceutically useful compositions comprising modulators of DmLGIC maybe formulated according to known methods such as by the admixture of apharmaceutically acceptable carrier. Examples of such carriers andmethods of formulation may be found in Remington's PharmaceuticalSciences. To form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the protein, DNA, RNA, modified DmLGIC, or either DmLGICagonists or antagonists including tyrosine kinase activators orinhibitors.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders.The effective amount may vary according to a variety of factors such asthe individual's condition, weight, sex and age. Other factors includethe mode of administration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents may be desirable.

The present invention also has the objective of providing suitabletopical, oral, systemic and parenteral pharmaceutical formulations foruse in the novel methods of treatment of the present invention. Thecompositions containing compounds identified according to this inventionas the active ingredient can be administered in a wide variety oftherapeutic dosage forms in conventional vehicles for administration.For example, the compounds can be administered in such oral dosage formsas tablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they mayalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The following examples are provided to illustrate the present inventionwithout, however, limiting the same hereto.

EXAMPLE 1 Isolation and Characterization of DNA Molecules EncodingDmLGIC

The molecular procedures were performed following standard procedureswell known in the art available in references such as Ausubel et. al.(1992, Short protocols in molecular biology. F. M. Ausubel etal.,—2^(nd.) ed. (John Wiley & Sons)) and Sambrook et al.(1989,Molecular cloning. A laboratory manual. J. Sambrook, E. F. Fritsch, andT. Maniatis—2^(nd) ed. (Cold Spring Harbor Laboratory Press)).

Ac05 and Ac15 Database Search—Partial sequences potentially encoding twonovel ligand gated ion channel genes, AC05 and AC15, were identified inthe Drosophila genome sequencing project using the Extended SmithWaterman algorithm. The query sequence was the C. elegans glutamategated ion channel avr-15a peptide sequence (accession number-AJ000538),and the DNA database searched was publicly available Drosophila highthroughput genomic sequences. The search was performed on a CompugenBiocel XLP hardware search engine (Petach Tikva, Israel).

Both sequences entered into the database contained predicted introns.Primers specific to either 05 or 15 were designed based on the databasesequences and synthesized. They are as follows: 1. ac05F1: 5′-CTT GCACAA AGC TGG CGT G-3′, [SEQ ID NO:8] (ac007805) 2. ac05F2: 5′-GTG AGC AGTATC GCA TAT TG-3′, [SEQ ID N0:9] (ac007805; 3. ac05R1: 5′-GTA GTT ATTTGA TAT GTC TAG C-3′, [SEQ ID NO:10] (ac007805); 4. ac05R2: 5′-ACC TGTTGA GTA CTC TAT AG-3′, [SEQ IDNO:11] (ac007805); 5. ac15F1: 5′-TTT GCACAG ACG TGG AAG G-3′, [SEQ ID NO:12] (ac007815); 6. ac15F2: 5′-ACA GGAATA CCG CCT GCT C-3′, [SEQ ID NO:13] (ac007815); and, 7. ac15R1: 5′-TTCATT TCG GAT GAG GGC CAC-3′ [SEQ ID NO:14] (ac007815);With these primer combinations, RT-PCR on whole fly total RNA followedby TA cloning was performed for both genes. Fragment of approximately500 bp for both Ac05 and Ac15 were isolated and verified by sequencing.

5′ and 3′ RACE for Ac05 and Ac15—The Marathon® cDNA Amplification Kitfrom Clontech (Palo Alto, Calif.) was used as the primary tool for both5′- and 3′-RACE reactions. PolyA⁺ RNA was purified from whole bodyOregon R Drosophila by Oligotex® mRNA Midi Kit (Qiagen, Santa Clarita,Calif.) and used to generate the double-stranded cDNA following themanufacturer's protocol. The following primers were used for RACEreactions:

3′-RACE Forward Primers:

Ac05:

-   1. Ac05GSPF1-5′-CAT CTT CCT TGC ACA AAG CTG GCG TG-3′ [SEQ ID    NO:15], (ac007805);-   2. Ac05NGSPF2-5′-CAT GAG TGA GCA GTA TCG CAT ATT G-3′ [SEQ ID    NO:16], (ac007805);    Ac15:-   1. Ac15GSPF1-5′-TGT GTT CTT TGC ACA GAC GTG GAA GG-3′ [SEQ ID NO:    17] (ac007815).-   2. Ac15NGSPF2 5′-TAT GAC ACA GGA ATA CCG CCT GCT C-3′ [SEQ ID NO:    18] (ac007815).    5′-RACE Reverse Primers:    Ac05:-   1. Ac05GSPR1: 5′-GTC TAG CTG CGG CAA CTC AAT CTC CGT G-3′ [SEQ ID    NO: 19], (ac007805);-   2. Ac05NGSPR2: 5′-CTC GAT CAT CAT GGA GCA GAT TTG CGT G-3′ [SEQ ID    NO:20], (ac007805).    Ac15:-   1. Ac15GSPR1: 5′-CGC CGT TTC ATT TCG GAT GAG GGC CAC-3′ [SEQ ID    NO:21], (ac007815 84958 bp - 84984 bp);-   2. Ac15NGSPR2: 5′-CAG GCT TTC CAT TTG CAG CTT GCA CTC C-3′ [SEQ ID    NO:22], (ac007815). This primer spans a splice junction, and the    existence of this continuous sequence was available only from the    sequence data of the Ac15 fragment described above.

5′ and 3′ RACE fragments were obtained for both genes by 1st round PCRand nested PCR based on the protocol of the Marathon® Kit, with amodification of the 5′RACE PCR cycle: 1 cycle of 1 minute at 94° C.; 5cycles of 1 minute at 94° C. and 4 minutes at 72° C.; and 25 cycles of 1minute at 94° C, 1 minutes at 68° C., and 3 minute at 72° C. Theresulting fragment sizes were ˜1.3 kb for Ac05 and ˜1.8 kb for Ac15 in3′-RACE. In 5′- RACE Ac05 and Ac15 both have fragment sizes of ˜1 kb.The PCR products were cloned into pCR2. 1-TOPO vector using the TOPO® TACloning Kit (Invitrogen, Carlsbad, Calif.). Miniprep DNA samples werescreened by restriction digestion to separate spliced from unsplicedclones. For the 3′ ends, 6 and 8 samples of Ac05 and Ac15, respectively,were sequenced. For the 5′ ends, 5 and 8 samples of Ac05 and Ac15 weresequenced.

Generation of Full-Length Clones—Using the sequences obtained from the5′ and 3′ RACE products, PCR primers for both genes were designed togenerate full-length clones. Forward primers and reverse primers forboth AcO5 and Ac15 were designed as follows: Ac05FullF1: [SEQ ID NO:23]5′-CAA TCG TCG CGA TAA CTC TGC CG-3′; Ac05FullR1: [SEQ ID NO:24] 5′-CCTTTA TTT ATA CAC TAC ATG GTA ATC-3′; +TL,32 Ac05FullR2: [SEQ ID NO:25]5′-TGT TTA CGC TCT ATT CCT TCG GAG-3′; Ac15FullF1: [SEQ ID NO:26] 5′-AACTGC CAA GAC GTT TAG AAC GG-3′; Ac15FullF2: [SEQ ID NO:27] 5′-CGA GTA AACTGT TAA ATG CTG AAG TG-3′; Ac15FullR1: [SEQ ID NO:28] 5′-TAC AAT TCA CTTAGG CTA CAT CAG C-3′; and, Ac15FullR2: [SEQ ID NO:29] 5′-GGC TAC ATC AGCTAC TAC GTC AC-3′.

The Advantage® 2 PCR Kit (Clonetech, Palo Alto, Calif.) was used for1^(st) and 2^(nd) round PCR. cDNA clones Ac05-10 and Ac05-11 weregenerated using primers Ac05 F1 and R1 for 1^(st) round PCR and primersAc05 F1 and R2 for 2^(nd) round PCR. cDNA clones Ac15-4 and Ac15-25 weregenerated using primers Ac15 F1 and R1 for 1^(st) round PCR. 1^(st)round PCR conditions were as follows: 1 cycle of 2 min at 94° C.; 5cycles of 30 sec at 94° C., and 2min 30sec at 72° C.; 25 cycles at 30sec at 94° C., 1 min at 68° C., and 1 min 30 sec at 72° C.; and 1 cycleat 5 min at 72° C. For ₂nd round PCR, primers Ac15 F1 and R2 were usedfor clone Ac15-4, and primers Ac15 F2 and R1 were used for cloneAc15-25. 2 μl of the first PCR product were used as template in a totalreaction volume of 50 μl. 2^(nd) round PCR conditions were as follows: 1cycle of 2 min at 94° C.; 5 cycles of 30 sec at 94° C., 1 min at 68° C.,and 1 min 30 sec at 72° C.; 5 cycles of 30 sec at 94° C., 1 min at 65°C., and 1 min 30 sec at 72° C.; 20 cycles of 30 sec at 94° C., 1 min at60° C., and 1 min 30sec at 72° C.; and 1 cycle of 5 min at 72° C. Onemajor band of ˜1.5 kb was isolated for Ac05 and ˜2 kb for Ac15. The PCRproducts were cloned into pCR2.1-TOPO vector using the TOPO® TA CloningKit (Invitrogene, Carlsbad, Calif.).

Two clones of Ac05 were identified: Ac05-10 (1518 bp) and Ac05-11 (1506bp). The clones are identical but for a 4 amino acid insertion withinthe M3-M4 intracellular loop in Ac05-10. Two clones of Ac15 have beenidentified: Ac15-4 (2073 bp) and Ac5-25 (2034 bp), which predict thesame protein sequence but differ in 16 nucleotides.

Synthesis of in vitro transcribed capped RNA—A PCR strategy was used toadd both the T7 promoter upstream of the initiating methionine (ATG),and a polyA⁺ tail following the stop codon (TGA and TAA for Ac05 andAc15) of the open reading frame (ORF) of clones Ac05-10, Ac05-11 andAc15-4, Ac15-25. The primers employed are: Ac05: Ac05T7: 5′-TAA TAC GACTCA CTA TAG GGA GGG [SEQ ID NO:30] TGT TCA TAA TGC AAA GCC-3′; and,Ac05dT, 5′-TTT TTT TTT TTT TTT TTT TTC ATA [SEQ ID NO:31] GGA ACG TTGTCC AAT AGA C-3′. Ac15: AC15T7: 5′-TAA TAC GAC TCA CTA TAG GGA GGC [SEQID NO:32] ACA TTA AAA TGG TGT TC-3′; and, AC15dT: 5′-TTT TTT TTT TTT TTTTTT TTC CTT [SEQ ID NO:33] ATA GAT ACT CGT AGA AC-3′.Amplified ORFs which contained both the T7 promoter and polyA⁺ tail werepurified using the Qiaquick PCR Purification Kit (Qiagen, Germany), andused directly as templates in the in vitro transcription reaction(mMessage mMachine™, Ambion, Austin, Tex.) following the manufacturer'sprotocol. After removal of the DNA template, the RNA was extracted withphenol/CHCl₃, precipitated with LiCl, and resuspended in nuclease-freewater at a storage concentration of 0.5 μg/μl.

EXAMPLE 2 Functional Expression of DmLGICs Clones in Xenopus oocytes

Full length cDNA clones corresponding to the selected RT-PCR sequenceswere used as template for synthesis of in vitro transcribed RNA (AmbionInc.). The capped cRNA transcripts are synthesized using appropriateoligonucleotide primers and the mMESSAGE mMACHINE in vitro RNAtranscription kit from Ambion. Xenopus laevis oocytes were prepared andinjected using standard methods as described (Arena et al., 1991, Mol.Pharmacol. 40: 368-374; Arena et al, 1992, Mol. Brain Res. 15: 339-348).Adult female Xenopus laevis were anesthetized with 0.17% tricainemethanesulfonate and the ovaries were surgically removed and placed in adish consisting of (mM): NaCl 82.5, KCl 2, MgCl₂ 1, CaCl₂ 1.8, HEPES 5,adjusted to pH 7.5 with NaOH (OR-2). Ovarian lobes were broken open,rinsed several times, and gently shaken in OR-2 containing 0.2%collagenase (Sigma, Type 1A) for 2-5 hours. When approximately 50% ofthe follicular layers were removed, Stage V and VI oocytes were selectedand placed in media consisting of (mM): NaCl 86, KCl 2, MgCl₂ 1, CaCl₂1.8, HEPES 5, Na pyruvate 2.5, theophylline 0.5, gentamicin 0.1 adjustedto pH 7.5 with NaOH (ND-96) for 24-48 hours before injection. For mostexperiments, oocytes were injected with 10 ng of cRNA in 50 nl of RNasefree water. Control oocytes were injected with 50 nl of water. Oocyteswere incubated for 1-5 days in ND-96 supplemented with 50 mg/mlgentamycin, 2.5 mM Na pyruvate and 0.5 mM theophylline before recording.Incubations and collagenase digestion were carried out at 18° C.

Voltage-clamp studies were conducted with the two microelectrode voltageclamp technique using a Dagan CA1 amplifier (Dagan Instruments,Minneapolis, Minn.). The current passing microelectrodes were filledwith 0.7 M KCl plus 1.7 M K₃-citrate and the voltage recordingmicroelectrodes were filled with 1.0 M KCl. The extracellular solutionfor most experiments was saline consisting of (mM): NaCl 96, BaCl₂ 3.5,MgCl₂ 0.5, CaCl₂ 0.1, HEPES 5, adjusted to pH 7.5 with NaOH. Theextracellular chloride concentration was reduced in some experiments byequimolar replacement of NaCl with the sodium salt of the indicatedanion. Experiments were conducted at 21-24° C. Data were acquired usingthe program Pulse and most analysis was performed with the companionprogram Pulsefit (Instrutech Instruments, Great Neck, N.Y.) or with IgorPro (Wavemetrics, Lake Oswego, Ore.). Data were filtered (f_(c), −3 db)at 1 kHz, unless otherwise indicated. FIG. 8 shows the results of theexperiment in which the clone DmLGIC AC05 clone was expressed in aXenopus oocyte. The measurement was made as described in this Examplewith the two microelectrode voltage clamp technique and the membranepotential was held at 0 mV. The bar at top shows the duration ofapplication of histamine. This indicates that expression of this proteinreconstitutes a functional ion channel that responds to the addition ofhistamine.

Expression of the AC15 clone in a Xenopus oocyte also forms a functionalsingle channel protein which, as with AC05, responds to the addition ofhistamine.

EXAMPLE 3 Functional Expression of DmLGICs Clones in Mammalian Cells

A DmLGIC may be subcloned into a mammalian expression vector and used totransfect the mammalian cell line of choice. Stable cell clones areselected by growth in the presence of G418. Single G418 resistant clonesare isolated and tested to confirm the presence of an intact DmLGICgene. Clones containing the DmLGICs are then analyzed for expressionusing immunological techniques, such as immuneprecipitation, Westernblot, and immunofluorescence using antibodies specific to the DmLGICproteins. Antibody is obtained from rabbits innoculated with peptidesthat are synthesized from the amino acid sequence predicted from theDmLGIC sequences. Expression is also analyzed using patch clampelectrophysiological techniques and an anion flux assay.

Cells that are expressing DmLGIC stably or transiently, are used to testfor expression of active channel proteins. These cells are used toidentify and examine other compounds for their ability to modulate,inhibit or activate the respective channel.

Cassettes containing the DmLGIC cDNA in the positive orientation withrespect to the promoter are ligated into appropriate restriction sites3′ of the promoter and identified by restriction site mapping and/orsequencing. These cDNA expression vectors may be introduced intofibroblastic host cells, for example, COS-7 (ATCC# CRL1651), and CV-1tat [Sackevitz et al.,1987, Science 238: 1575], 293, L (ATCC# CRL6362)by standard methods including but not limited to electroporation, orchemical procedures (cationic liposomes, DEAE dextran, calciumphosphate). Transfected cells and cell culture supernatants can beharvested and analyzed for DmLGIC expression as described herein.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing DmLGIC. Unaltered DmLGIC cDNAconstructs cloned into expression vectors are expected to program hostcells to make DmLGIC protein. In addition, DmLGIC is expressedextracellularly as a secreted protein by ligating DmLGIC cDNA constructsto DNA encoding the signal sequence of a secreted protein. Thetransfection host cells include, but are not limited to, CV-1-P[Sackevitz et al.,1987, Science 238: 1575], tk-L [Wigler, et al., 1977,Cell 11: 223 ], NS/0, and dHFr-CHO [Kaufman and Sharp, 1982, J. Mol.Biol. 159: 601].

Co-transfection of any vector containing a DmLGIC cDNA with a drugselection plasmid including, but not limited to G418, aminoglycosidephosphotransferase; hygromycin, hygromycin-B phosphotransferase; APRT,xanthine-guanine phosphoribosyl-transferase, will allow for theselection of stably transfected clones. Levels of DmLGIC are quantitatedby the assays described herein. DmLGIC cDNA constructs may also beligated into vectors containing amplifiable drug-resistance markers forthe production of mammalian cell clones synthesizing the highestpossible levels of DmLGIC. Following introduction of these constructsinto cells, clones containing the plasmid are selected with theappropriate agent, and isolation of an over-expressing clone with a highcopy number of plasmids is accomplished by selection with increasingdoses of the agent. The expression of recombinant DmLGIC is achieved bytransfection of full-length DmLGIC cDNA into a mammalian host cell.

EXAMPLE 4 Cloning of DmLGIC cDNA into a Baculovirus Expression Vectorfor Expression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL# 1711). A recombinant baculoviruseexpressing DmLGIC cDNA is produced by the following standard methods(InVitrogen Maxbac Manual): the DmLGIC cDNA constructs are ligated intothe polyhedrin gene in a variety of baculovirus transfer vectors,including the pAC360 and the BlueBac vector (InVitrogen). Recombinantbaculoviruses are generated by homologous recombination followingco-transfection of the baculovirus transfer vector and linearized AcNPVgenomic DNA [Kitts, 1990, Nuc. Acid. Res. 18: 5667] into Sf9-cells.Recombinant pAC360 viruses are identified by the absence of inclusionbodies in infected cells and recombinant pBlueBac viruses are identifiedon the basis of b-galactosidase expression (Summers, M. D. and Smith, G.E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaquepurification, DmLGIC expression is measured by the assays describedherein.

The cDNA encoding the entire open reading frame for DmLGIC is insertedinto the BamHI site of pBlueBacII. Constructs in the positiveorientation are identified by sequence analysis and used to transfectSf9 cells in the presence of linear AcNPV mild type DNA.

Authentic, active DmLGIC is found in the cytoplasm of infected cells.Active DmLGIC is extracted from infected cells by hypotonic or detergentlysis.

EXAMPLE 5 Cloning of DmLGIC cDNA into a Yeast Expression Vector

Recombinant DmLGIC is produced in the yeast S. cerevisiae following theinsertion of the optimal DmLGIC cDNA cistron into expression vectorsdesigned to direct the intracellular or extracellular expression ofheterologous proteins. In the case of intracellular expression, vectorssuch as EmBLyex4 or the like are ligated to the DmLGIC cistron [Rinas,et al., 1990, Biotechnology 8: 543-545; Horowitz B. et al., 1989, J.Biol. Chem. 265: 4189-4192]. For extracellular expression, the DmLGICcistron is ligated into yeast expression vectors which fuse a secretionsignal (a yeast or mammalian peptide) to the NH₂ terminus of the DmLGICprotein [Jacobson, 1989, Gene 85: 511-516; Riett and Bellon, 1989,Biochem. 28: 2941-2949].

These vectors include, but are not limited to pAVE1-6, which fuses thehuman serum albumin signal to the expressed cDNA [Steep, 1990,Biotechnology 8: 42-46], and the vector pL8PL which fuses the humanlysozyme signal to the expressed cDNA [Yamamoto, Biochem. 28:2728-2732)]. In addition, DmLGIC is expressed in yeast as a fusionprotein conjugated to ubiquitin utilizing the vector pVEP [Ecker, 1989,J. Biol. Chem. 264: 7715-7719, Sabin, 1989 Biotechnology 7: 705-709,McDonnell, 1989, Mol. Cell Biol. 9: 5517-5523 (1989)]. The levels ofexpressed DmLGIC are determined by the assays described herein.

EXAMPLE 6 Purification of Recombinant DmLGIC

Recombinantly produced DmLGIC may be purified by antibody affinitychromatography. DmLGIC antibody affinity columns are made by adding theanti-DmLGIC antibodies to Affigel-10 (Biorad), a gel support which ispre-activated with N-hydroxysuccinimide esters such that the antibodiesform covalent linkages with the agarose gel bead support. The antibodiesare then coupled to the gel via amide bonds with the spacer arm. Theremaining activated esters are then quenched with 1M ethanolamine HCl(pH 8). The column is washed with water followed by 0.23 M glycine HCl(pH 2.6) to remove any non-conjugated antibody or extraneous protein.The column is then equilibrated in phosphate buffered saline (pH 7.3)together with appropriate membrane solubilizing agents such asdetergents and the cell culture supernatants or cell extracts containingsolubilized DmLGIC are slowly passed through the column. The column isthen washed with phosphate-buffered saline together with detergentsuntil the optical density (A280) falls to background, then the proteinis eluted with 0.23 M glycine-HCl (pH 2.6) together with detergents. Thepurified DmLGIC protein is then dialyzed against phosphate bufferedsaline.

EXAMPLE 7 Purification of Recombinant DmLGIC

According to the Drosophila genome sequencing project, Ac007815 [Ac15]and Ac007805 [Ac05] map to chromosome III; specifically Ac15 to ChIII92B and Ac05 to ChIII 87B. DrosGluClalpha1 (glc-1), maps to ChIII 92B,as does Ac15. O'Tousa et al. (1989, J. Neurogenetics 6: 41-52) mapphotoreceptor mutations to the ChIII 92B region of the Droshphilagenome. In addition, Stuart (1999, Neuron 22:431433) notes thathistamine is a potential invertebrate retinal neurotransmitter.Therefore, the data generated in Example section 2 herein in combinationwith the chromosomal location of Ac15 suggests that the LGIC disclosedherein are at least partially effective as being responsive tohistamine.

1. A purified nucleic acid molecule encoding a Drosophila LGIC protein,wherein said protein comprises an amino acid sequence as set forth inSEQ ID NO:2.
 2. An expression vector for expressing a Drosophila LGICprotein in a recombinant host cell wherein said expression vectorcomprises a DNA molecule of claim
 1. 3. A host cell which expresses arecombinant Drosophila LGIC protein wherein said host cell contains theexpression vector of claim
 2. 4. A process for expressing a DrosophilaLGIC protein in a recombinant host cell, comprising: (a) transfectingthe expression vector of claim 3 into a suitable host cell; and, (b)culturing the host cells of step (a) under conditions which allowexpression of said Drosophila LGIC protein from said expression vector.5. A purified DNA molecule encoding a Drosophila LGIC protein whichcomprises a nucleotide sequence as set forth in SEQ ID NO:
 1. 6. The DNAmolecule of claim 5 containing from about nucleotide 199 to aboutnucleotide 1479 of SEQ ID NO: 1
 7. A purified nucleic acid moleculeencoding a Drosophila LGIC protein, wherein said protein comprises anamino acid sequence as set forth in SEQ ID NO:4.
 8. An expression vectorfor expressing a Drosophila LGIC protein in a recombinant host cellwherein said expression vector comprises a DNA molecule of claim
 7. 9. Ahost cell which expresses a recombinant Drosophila LGIC protein whereinsaid host cell contains the expression vector of claim
 8. 10. A processfor expressing a Drosophila LGIC protein in a recombinant host cell,comprising: (a) transfecting the expression vector of claim 9 into asuitable host cell; and, (b) culturing the host cells of step (a) underconditions which allow expression of said Drosophila LGIC protein fromsaid expression vector.
 11. A purified DNA molecule encoding aDrosophila LGIC protein which comprises a nucleotide sequence as setforth in SEQ ID NO:3.
 12. The DNA molecule of claim 11 containing fromabout nucleotide 199 to about nucleotide 1467 of SEQ ID NO:3.
 13. Apurified nucleic acid molecule encoding a Drosophila LGIC protein,wherein said protein comprises an amino acid sequence as set forth inSEQ ID NO:7.
 14. An expression vector for expressing a Drosophila LGICprotein in a recombinant host cell wherein said expression vectorcomprises a DNA molecule of claim
 13. 15. A host cell which expresses arecombinant Drosophila LGIC protein wherein said host cell contains theexpression vector of claim
 14. 16. A process for expressing a DrosophilaLGIC protein in a recombinant host cell, comprising: (a) transfectingthe expression vector of claim 15 into a suitable host cell; and, (b)culturing the host cells of step (a) under conditions which allowexpression of said Drosophila LGIC protein from said expression vector.17. A purified DNA molecule encoding a Drosophila LGIC protein whichcomprises a nucleotide sequence selected from the group consisting ofnucleotide sequences as set forth in SEQ ID NO:5 and SEQ ID NO:6. 18.The DNA molecule of claim 17 containing from about nucleotide 330 toabout nucleotide 1787 of SEQ ID NO:3.
 19. The DNA molecule of claim 17containing from about nucleotide 278 to about nucleotide 1735 of SEQ IDNO:3.
 20. A Drosophila LGIC protein substantially free from otherproteins which comprises an amino acid sequence as set forth in SEQ IDNO:2.
 21. A Drosophila LGIC protein of claim 20 which is a product of aDNA expression vector contained within a recombinant host cell.
 22. Asubstantially pure membrane preparation comprising the Drosophila LGICprotein purified from the recombinant host cell of claim
 21. 23. ADrosophila LGIC protein substantially free from other proteins whichcomprises an amino acid sequence as set forth in SEQ ID NO:4.
 24. ADrosophila LGIC protein of claim 23 which is a product of a DNAexpression vector contained within a recombinant host cell.
 25. Asubstantially pure membrane preparation comprising the Drosophila LGICprotein purified from the recombinant host cell of claim
 24. 26. ADrosophila LGIC protein substantially free from other proteins whichcomprises an amino acid sequence as set forth in SEQ ID NO:7.
 27. ADrosophila LGIC protein of claim 26 which is a product of a DNAexpression vector contained within a recombinant host cell.
 28. Asubstantially pure membrane preparation comprising the Drosophila LGICprotein purified from the recombinant host cell of claim
 27. 29. ADrosophila LGIC protein which consists of an amino acid sequenceselected from the group consisting of amino acid sequences as set forthin SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:7.
 30. A Drosophila LGIChomomultimer channel receptor substantially free from other proteinswhich comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7.
 31. A DrosophilaLGIC protein of claim 30 which is a product of a DNA expression vectorcontained within a recombinant host cell.
 32. A substantially puremembrane preparation comprising the Drosophila LGIC channel purifiedfrom the recombinant host cell of claim
 31. 33. A Drosophila LGICheteromultimer channel receptor substantially free from other proteinswhich comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7.
 34. A DrosophilaLGIC protein of claim 33 which is a product of a DNA expression vectorcontained within a recombinant host cell.
 35. A substantially puremembrane preparation comprising the Drosophila LGIC channel purifiedfrom the recombinant host cell of claim
 34. 36. A method of identifyinga modulator of a LGIC protein, comprising: (a) contacting a testcompound with a Drosophila LGIC protein selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7; and, (b)measuring the effect of the test compound on the LGIC protein.
 37. Themethod of claim 36 wherein the Drosophila LGIC protein of step (a) is aproduct of a DNA expression vector contained within a recombinant hostcell.
 38. A method of identifying a compound that modulatesglutamate-gated channel protein activity, which comprises: a) injectinginto a host cell solution a population of nucleic acid molecules, atleast of portion of which encodes a Drosophila LGIC protein selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7,such that expression of said portion of nucleic acid molecules resultsin an active glutamate-gated channel; and, b) adding a test compoundinto said solution; c) measuring host cell membrane current at a holdingpotential more positive than the reversal potential for chloride. 39.The method of claim 38 wherein said nucleic acid molecule is selectedfrom the group consisting of complementary DNA, poly A⁺ messenger RNAand complementary RNA.